Enzyme-pore constructs

ABSTRACT

The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/858,138, filed Sep. 18, 2015, which is a divisional of U.S. patentapplication Ser. No. 14/455,394 filed Aug. 8, 2014, now U.S. Pat. No.9,885,078, which is a divisional of U.S. patent application Ser. No.13/002,709, filed May 13, 2011, now abandoned, which is a 35 U.S.C. 371national stage filing of International Application No. PCT/GB2009/001679filed Jul. 6, 2009, which claims the benefit under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 61/078,695 filed Jul. 7, 2008.The contents of the above referenced applications are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to constructs comprising a transmembrane proteinpore subunit and a nucleic acid handling enzyme. The pore subunit iscovalently attached to the enzyme such that both the subunit and enzymeretain their activity. The constructs can be used to generatetransmembrane protein pores having a nucleic acid handling enzymeattached thereto. Such pores are particularly useful for sequencingnucleic acids. The enzyme handles the nucleic acid in such a way thatthe pore can detect each of its component nucleotides by stochasticsensing.

BACKGROUND OF THE INVENTION

Stochastic detection is an approach to sensing that relies on theobservation of individual binding events between analyte molecules and areceptor. Stochastic sensors can be created by placing a single pore ofnanometer dimensions in an insulating membrane and measuringvoltage-driven ionic transport through the pore in the presence ofanalyte molecules. The frequency of occurrence of fluctuations in thecurrent reveals the concentration of an analyte that binds within thepore. The identity of an analyte is revealed through its distinctivecurrent signature, notably the duration and extent of current block(Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L.,Gouaux, J. E., and Bayley, H. (1997) Chem. Biol. 4, 497-505; and Bayley,H., and Cremer, P. S. (2001) Nature 413, 226-230).

Engineered versions of the bacterial pore forming toxin α-hemolysin(α-HL) have been used for stochastic sensing of many classes ofmolecules (Bayley, H., and Cremer, P. S. (2001) Nature 413, 226-230;Shin, S., H., Luchian. T., Cheley, S., Braha, P., and Bayley, H. (2002)Angew. Chem. Int. Ed. 41, 3707-3709; and Guan, X., Gu, L.-Q., Cheley,S., Braha, O., and Bayley, H. (2005) ChemBioChem 6, 1875-1881). In thecourse of these studies, it was found that attempts to engineer α-HL tobind small organic analytes directly can prove taxing, with rareexamples of success (Guan, X., Gu. L.-Q., Cheley, S., Braha, O., andBayley, H. (2005) ChemBioChem 6, 1875-1881). Fortunately, a differentstrategy was discovered, which utilized non-covalently attachedmolecular adaptors, notably cyclodextrins (Gu, L.-Q., Braha, O., Conlan,S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690), but alsocyclic peptides (Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H., andBraha, O. (2000) J. Am. Chem. Soc. 122, 11758-11766) and cucurbiturils(Braha. O., Webb, J., Gu, L.-Q., Kim, K., and Bayley, H. (2005)ChemPhysChem 6, 889-892). Cyclodextrins become transiently lodged in theα-HL pore and produce a substantial but incomplete channel block.Organic analytes, which bind within the hydrophobic interiors ofcyclodextrins, augment this block allowing analyte detection (Gu, L.-Q.,Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999) Nature 398,686-690).

There is currently a need for rapid and cheap DNA or RNA sequencingtechnologies across a wide range of applications. Existing technologiesare slow and expensive mainly because they rely on amplificationtechniques to produce large volumes of nucleic acid and require a highquantity of specialist fluorescent chemicals for signal detection.Stochastic sensing has the potential to provide rapid and cheap DNAsequencing by reducing the quantity of nucleotide and reagents required.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that covalent attachment ofa transmembrane protein pore subunit to a nucleic acid handling enzymeresults in a construct that is capable of both forming a pore andhandling nucleic acids. The inventors have also surprisinglydemonstrated that the construct can be used to generate a transmembraneprotein pore that is capable of both handling a nucleic acid andsequencing the nucleic acid via stochastic sensing. The fixed nature andclose proximity of the enzyme to the pore means that a proportion of thenucleotides in a target nucleic acid will interact with the pore andaffect the current flowing through the pore in a distinctive manner. Asa result, transmembrane protein pores comprising such constructs areuseful tools for stochastic sensing and especially for sequencingnucleic acids.

Accordingly, the invention provides a construct comprising atransmembrane protein pore subunit and a nucleic acid handling enzyme,wherein the subunit is covalently attached to the enzyme, wherein thesubunit retains its ability to form a pore and wherein the enzymeretains its ability to handle nucleic acids. The invention alsoprovides:

-   -   a polynucleotide sequence which encodes a construct of the        invention;    -   a modified pore for use in sequencing nucleic acids, comprising        at least one construct of the invention;    -   a kit for producing a modified pore for use in sequencing        nucleic acids, comprising:    -   (a) at least one construct of the invention; and    -   (b) any remaining subunits needed to form a pore;    -   a kit for producing a modified pore for use in sequencing        nucleic acids, comprising:    -   (b) at least one polynucleotide of the invention; and    -   (c) polynucleotide sequences encoding any remaining subunits        needed to form a pore;    -   a method of producing a construct of the invention, comprising:    -   (a) covalently attaching a nucleic acid handling enzyme to a        transmembrane protein pore subunit; and    -   (b) determining whether or not the resulting construct is        capable of forming a pore and handling nucleic acids;    -   a method of producing a modified pore of the invention,        comprising:    -   (a) covalently attaching a nucleic acid handling enzyme to a        transmembrane protein pore; and    -   (b) determining whether or not the resulting pore is capable of        handling nucleic acids and detecting nucleotides;    -   method of producing a modified pore of the invention,        comprising:    -   (a) allowing at least one construct of the invention to form a        pore with other suitable subunits; and    -   (b) determining whether or not the resulting pore is capable of        handling nucleic acids and detecting nucleotides.    -   a method of purifying a transmembrane pore comprising at least        one construct of the invention, comprising:    -   (a) providing the at least one construct and the other subunits        required to form the pore:    -   (b) oligomerising the at least one construct and other subunits        on synthetic lipid vesicles; and    -   (c) contacting the vesicles with a non-ionic surfactant; and    -   (d) recovering the oligomerised pore;    -   a method of sequencing a target nucleic acid sequence,        comprising:    -   (a) contacting the target sequence with a pore of the invention,        which comprises an exonuclease, such that the exonuclease        digests an individual nucleotide from one end of the target        sequence;    -   (b) contacting the nucleotide with the pore so that the        nucleotide interacts with the adaptor;    -   (c) measuring the current passing through the pore during the        interaction and thereby determining the identity of the        nucleotide; and    -   (d) repeating steps (a) to (c) at the same end of the target        sequence and thereby determining the sequence of the target        sequence; and    -   a method of sequencing a target nucleic acid sequence,        comprising:    -   (a) contacting the target sequence with a pore of the invention        so that the enzyme pushes or pulls the target sequence through        the pore and a proportion of the nucleotides in the target        sequence interacts with the pore; and    -   (b) measuring the current passing through the pore during each        interaction and thereby determining the sequence of the target        sequence.

DESCRIPTION OF THE FIGURES

FIG. 1 shows how exonuclease enzymes catalyse the hydrolysis ofphosphodiester bonds. Within the active site of the exonuclease, a watermolecule is enabled to react with the phosphate of the 3′ end of thepolynucleotide (DNA). Cleavage of the bond between the phosphate and thesugar towards the 5′ end releases a monophosphate (deoxy)nucleoside.

FIG. 2 shows the crystal structures of exonucleases used in the Example,N and C-terminus and active sites are shown for each. i) Adapted form ofEcoExoIII; ii) EcoExoI; iii) TthRecJ-cd; and iv) Lambda exo.

FIG. 3 shows a cartoon of an exonuclease equipped α-HL pore. Theexonuclease is genetically fused to one of the seven monomers of theheptamer, with linker arms sufficiently long to enable correct proteinfolding of the exonuclease moiety and the α-HL moiety.

FIG. 4 shows generic image of the protein construct generated shows theBspEI insertion point(s) in the α-HL gene. Ligation AfuExoIII, boundedby two stretches of DNA encoding a (serine/glycine)×5 repeat (shownhatched) generates a fusion protein in which a 64.5 kDa protein will begenerated, under the transcriptional control of the T7 promoter shown.

FIG. 5 shows the oligomerisation of α-HL Loop 1 fusion constructs withwild-type α-HL at different protein ratios. i) HL-wt-EcoExoIII-L1-H6;ii) HL-RQC-EcoExoI-L1-H6; and iii) HL-RQC-TthRecJ-L1-H6.

FIG. 6 shows the control of homo and heteroheptamer generation bydifferent monomer ratios. HL-RQ subunits are shown in white and fusionsubunits in black. Increasing the ratio of fusion subunits to wild-typesubunits increases the generation of 2:5, 1:6 and 0:7 hetero andhomo-heptamers. Similarly increasing the concentration of HL-RQ monomerincreases the generation of 6:1 and 5:2 heteroheptamers.

FIG. 7 shows the oligomerisation of HL-RQC-EcoExoIII-L1-H6 fusionproteins that contain a stiff polyproline EcoExoIII C-terminus linker.IVTT expressed proteins mixed in a 5:1 wild-type to fusion protein ratioin the presence of purified rabbit red blood cell membranes. i)HL-RQC-EcoExoIII-L1-{SG}5+{SG}5-H6; ii) HL-RQC-EcoExoIII-L1-{SG}5+5P-H6;iii) HL-RQC-EcoExoIII-L1-4SG+5P-H6; and iv) HL monomers.

FIG. 8 shows the Loop 2 region of a single α-hemolysin subunit with themature heptamer. Subunit 1 shown in white, subunits 2-7 shown in greyand the loop 2 region of subunit 1 shown in black.

FIG. 9 shows the oligomerisation of alternative Loop 2 EcoExoIII fusionproteins. i) HL-(RQ)₇; ii) HL-(RQ)₆(RQC-EcoExoIII-L2a-H6)₁; iii)HL-(RQ)₆(RQC-EcoExoIII-L2a-8P-H6)₁; iv)HL-(RQ)₆(RQC-EcoExoIII-L2-H48Δ-H6)₁; v)HL-(RQ)₆(RQC-EcoExoIII-L2-D45Δ-H6)₁; vi)HL-(RQ)₆(RQC-EcoExoIII-L2-D45-K46Δ-H6)₁; and vii)HL-(RQ)₆(RQC-EcoExoIII-L2-D45-N47Δ-H6)₁.

FIG. 10 shows the oligomerisation of alternative Loop 2 EcoExoIII fusionproteins. i) HL-(RQ)₇; ii) HL-(RQ)₆(RQC-EcoExoIII-L2a-H6)₁; iii)HL-(RQ)₆(RQC-EcoExoIII-L2-D45-N47Δ-H6)₁; iv)HL-(RQ)₆(RQC-EcoExoIII-L2-D46-K56Δ-H6)₁; v)HL-(RQ)₆(RQC-EcoExoIII-L2-D46Δ-H6)₁; vi)HL-(RQ)₆(RQC-EcoExoIII-L2-D46-N47Δ-H6)₁; vii)HL-(RQ)₆(RQC-EcoExoIII-L2-A1-S16Δ/D46-N47Δ-H6)₁; viii)HL-(RQ)₆(RQC-EcoExoIII-L2-F42-D46Δ-H6)₁; and ix)HL-(RQ)₆(RQC-EcoExoIII-L2-143-D46Δ-H6)₁.

FIG. 11 shows the oligomerisation of EcoExoI C-terminus fusion proteins.a) denotes both hemolysin and enzyme-fusion protein monomers areradiolabelled, b) denotes only the fusion protein monomer isradiolabelled. i) HL-(RQ)₆(RQC-EcoExoI-Cter-{SG}8-H6)₁; ii)HL-(RQ)₆(RQC-EcoExoI-Cter-DG{SG}8-H6)₁; iii)HL-(RQ)₆(RQC-EcoExoI-Cter-WPV{SG}8-H6)₁; iv)HL-(RQ)₆(RQC-EcoExoI-Cter-DGS{P}12-H6)₁; and v)HL-(RQ)₆(RQC-EcoExoI-Cter-WPV{P}12-H6)₁.

FIGS. 12A and 12B show the effect of different surfactants on EcoExoIIIactivity. Bottom graph (FIG. 12B)—Sodium dodecyl sulphate (SDS): a; 0%,b; 0.1%, c; 0.5%. Top graph (FIG. 12A)—n-Dodecyl-D-maltopyranoside(DDM): a; 0%, b; 0.1%, c; 0.25%, d; 0.5%.

FIG. 13 shows the oligomerisation of E. coli BL21 (DE3) pLysS expressedα-hemolysin monomers for formation and purification of preferentially6:1 heteroheptamers. His-tag purification is used to select betweenheteroheptamers and wild-type homoheptamer to give a large excess of 6:1heteroheptamer.

FIG. 14 shows the exonuclease activity of monomer and heteroheptamerfusion proteins. Left graph—Activity of Wild-type and fusion monomers:a, 10^(−′2) dilution HL-RQC-EcoExoIII-L1-H6; b, 10^(−′4) dilutionHL-RQC-EcoExoIII-L1-H6; c, 10^(−′6) dilution HL-RQC-EcoExoIII-L1-H6; d,10^(−′2) dilution HL-RQ. Right graph—Activity ofHL-(RQ)₆(RQC-EcoExoIII-L1-H6)₁: a, DDM crude extract; b. Ni-NTApurified; c. Ni-NTA purified and buffer exchange.

FIG. 15 shows base detection by theHL-(RQ)₆(RQC-EcoExoIII-L2-D46-N47Δ-H6) heteroheptamer. The top trace wasobtained from a heteroheptamer with a covalently attached am₆-amPDP₁-βCDadapter molecule. Further blocking events can be seen and ascribed toindividual mono-phosphate nucleosides for base discrimination. Thebottom graph shows the corresponding histograms of dNMP events from thetop trace. Peaks, from left to right, correspond to G, T, A, Crespectively. Data acquired at 400/400 mM KCl, 180 mV and 10 μM dNMPs.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the polynucleotide sequence encoding one subunit ofwild-type α-hemolysin (α-HL).

SEQ ID NO: 2 shows the amino acid sequence of one subunit of wild-typeα-HL. Amino acids 2 to 6, 73 to 75, 207 to 209, 214 to 216 and 219 to222 form α-helices. Amino acids 22 to 30, 35 to 44, 52 to 62, 67 to 71,76 to 91, 98 to 103, 112 to 123, 137 to 148, 154 to 159, 165 to 172, 229to 235, 243 to 261, 266 to 271, 285 to 286 and 291 to 293 formβ-strands. All the other non-terminal amino acids, namely 7 to 21, 31 to34, 45 to 51.63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153,160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242,262 to 265, 272 to 274 and 287 to 290 form loop regions. Amino acids 1and 294 are terminal amino acids.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit ofα-HL M113R/N139Q (HL-RQ).

SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HLM113R/N139Q (HL-RQ). The same amino acids that form α-helices, β-strandsand loop regions in wild-type α-HL form the corresponding regions inthis subunit.

SEQ ID NO: 5 shows the pT7 α-HL BspEI knockout polynucleotide sequence(pT7-SC1_BspEI-KO). The α-HL encoding sequence is between nucleotides2709 and 3593. The BspEI remnant is at nucleotides 3781 and 3782.

SEQ ID NO: 6 shows the polynucleotide sequence encoding one subunit ofwild-type α-hemolysin containing a BspEI cloning site at position 1(L1).

SEQ ID NO: 7 shows the polynucleotide sequence encoding one subunit ofwild-type α-hemolysin containing a BspEI cloning site at position 2(L2a).

SEQ ID NO: 8 shows the polynucleotide sequence encoding one subunit ofwild-type α-hemolysin containing a BspEI cloning site at position 2(L2b).

SEQ ID NO: 9 shows the codon optimized polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 10 shows the amino acid sequence of the exonuclease II enzymefrom E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides. Amino acids 11 to 13, 15 to 25,39 to 41, 44 to 49, 85 to 89, 121 to 139, 158 to 160, 165 to 174, 181 to194, 198 to 202, 219 to 222, 235 to 240 and 248 to 252 form α-helices.Amino acids 2 to 7, 29 to 33, 53 to 57, 65 to 70, 75 to 78, 91 to 98,101 to 109, 146 to 151, 195 to 197, 229 to 234 and 241 to 246 form1-strands. All the other non-terminal amino acids, 8 to 10, 26 to 28, 34to 38, 42, 43, 50 to 52, 58 to 64, 71 to 74, 79 to 84, 90, 99, 100, 110to 120, 140 to 145, 152 to 157, 161 to 164, 175 to 180, 203 to 218, 223to 228, 247 and 253 to 261, form loops. Amino acids 1, 267 and 268 areterminal amino acids. The enzyme active site is formed by loop regionsconnecting β₁-α₁, β₃-β₄, β₅-β₆, β_(III)-α_(I), β_(IV)-α_(II) andβ_(V)-β_(VI) (consisting of amino acids 8-10, 58-64, 90, 110-120,152-164, 175-180, 223-228 and 253-261 respectively). A single divalentmetal ion is bound at residue E34 and aids nucleophilic attack on thephosphodiester bond by the D229 and H259 histidine-aspartate catalyticpair.

SEQ ID NO: 11 shows the codon optimized polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExoI from E. coli.

SEQ ID NO: 12 shows the amino acid sequence of exonuclease I enzyme(EcoExoI) from E. coli. This enzyme performs processive digestion of 5′monophosphate nucleosides from single stranded DNA (ssDNA) in a 3′-5′direction. Enzyme initiation on a strand requires at least 12nucleotides. Amino acids 60 to 68, 70 to 78, 80 to 93, 107 to 119, 124to 128, 137 to 148, 165 to 172, 182 to 211, 213 to 221, 234 to 241, 268to 286, 313 to 324, 326 to 352, 362 to 370, 373 to 391, 401 to 454 and457 to 475 form α-helices. Amino acids 10 to 18, 28 to 26, 47 to 50, 97to 101, 133 to 136, 229 to 232, 243 to 251, 258 to 263, 298 to 302 and308 to 311 form β-strands. All the other non-terminal amino acids, 19 to27, 37 to 46, 51 to 59, 69, 79, 94 to 96102 to 106, 120 to 123, 129 to132, 149 to 164, 173 to 181, 212, 222 to 228 233, 242, 252 to 257, 264to 267, 287 to 297, 303 to 307, 312, 325, 353 to 361, 371, 372, 392 to400455 and 456, form loops. Amino acids 1 to 9 are terminal amino acids.The overall fold of the enzyme is such that three regions combine toform a molecule with the appearance of the letter C, although residues355-358, disordered in the crystal structure, effectively convert this Cinto an O-like shape. The amino terminus (1-206) forms the exonucleasedomain and has homology to the DnaQ superfamily, the following residues(202-354) form an SH3-like domain and the carboxyl domain (359-475)extends the exonuclease domain to form the C-like shape of the molecule.Four acidic residues of EcoExoI are conserved with the active siteresidues of the DnaQ superfamily (corresponding to D15, E17, D108 andD186). It is suggested a single metal ion is bound by residues D15 and108. Hydrolysis of DNA is likely catalyzed by attack of the scissilephosphate with an activated water molecule, with H181 being thecatalytic residue and aligning the nucleotide substrate.

SEQ ID NO: 13 shows the codon optimized polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 14 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides. Amino acids 19to 33, 44 to 61, 80 to 89, 103 to 111, 136 to 140, 148 to 163, 169 to183, 189 to 202, 207 to 217, 223 to 240, 242 to 252, 254 to 287, 302 to318, 338 to 350 and 365 to 382 form α-helices. Amino acids 36 to 40, 64to 68, 93 to 96, 116 to 120, 133 to 135, 294 to 297, 321 to 325, 328 to332, 352 to 355 and 359 to 363 form β-strands. All the othernon-terminal amino acids, 34, 35, 41 to 43, 62, 63, 69 to 79, 90 to 92,97 to 102, 112 to 115, 121 to 132, 141 to 147, 164 to 168, 184 to 188203to 206, 218 to 222, 241, 253, 288 to 293, 298 to 301, 319, 320, 326,327, 333 to 337, 351 to 358 and 364, form loops. Amino acids 1 to 18 and383 to 425 are terminal amino acids. The crystal structure has only beenresolved for the core domain of RecJ from Thermus thermophilus (residues40-463). To ensure initiation of translation and in vivo expression ofthe RecJ core domain a methionine residue was added at its aminoterminus, this is absent from the crystal structure information. Theresolved structure shows two domains, an amino (2-253) and a carboxyl(288-463) region, connected by a long α-helix (254-287). The catalyticresidues (D46, D98, H122, and D183) co-ordinate a single divalent metalion for nucleophilic attack on the phosphodiester bond. D46 and H120proposed to be the catalytic pair, however, mutation of any of theseconserved residues in the E. coli RecJ was shown to abolish activity.

SEQ ID NO: 15 shows the codon optimized polynucleotide sequence derivedfrom the bacteriphage lambda exo (redX) gene. It encodes thebacteriphage lambda exonuclease.

SEQ ID NO: 16 shows the amino acid sequence of the bacteriphage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 3′-5′ direction. Enzymeinitiation on a strand preferentially requires a 5′ overhang ofapproximately 4 nucleotides with a 5′ phosphate. Amino acids 3 to 10, 14to 16, 22 to 26, 34 to 40, 52 to 67, 75 to 95, 135 to 149, 152 to 165and 193 to 216 form α-helices. Amino acids 100 to 101, 106 to 107, 114to 116, 120 to 122, 127 to 131, 169 to 175 and 184 to 190 form3-strands. All the other non-terminal amino acids, 11 to 13, 17 to 21,27 to 33, 41 to 51, 68 to 74, 96 to 99, 102 to 105, 108 to 113, 117 to119, 123 to 126, 132 to 134, 150 to 151, 166 to 168, 176 to 183, 191 to192, 217 to 222, form loops. Amino acids 1, 2 and 226 are terminal aminoacids. Lambda exonuclease is a homo-trimer that forms a toroid with atapered channel through the middle, apparently large enough for dsDNA toenter at one end and only ssDNA to exit at the other. The catalyticresidues are undetermined but a single divalent metal ion appears boundat each subunit by residues D119, E129 and L130.

SEQ ID NO: 17 shows the polynucleotide sequence encodingHL-wt-EcoExoIII-L1-H6 used in the Example.

SEQ ID NO: 18 shows the amino acid sequence of one subunit ofHL-wt-EcoExoIII-L1-H6 used in the Example.

SEQ ID NO: 19 shows the polynucleotide sequence encodingHL-RQC-EcoExoIII-L1-H6 used in the Example.

SEQ ID NO: 20 shows the amino acid sequence of one subunit ofHL-RQC-EcoExoIII-L1-H6 used in the Example.

SEQ ID NO: 21 shows the polynucleotide sequence encodingHL-RQC-EcoExoI-L1-H6 used in the Example.

SEQ ID NO: 22 shows the amino acid sequence of one subunit ofHL-RQC-EcoExoI-L1-H6 used in the Example.

SEQ ID NO: 23 shows the polynucleotide sequence encodingHL-RQC-TthRecJ-L1-H6 used in the Example.

SEQ ID NO: 24 shows the amino acid sequence of one subunit ofHL-RQC-TthRecJ-L1-H6 used in the Example.

SEQ ID NO: 25 shows the polynucleotide sequence encodingHL-RQC-EcoExoIII-L2-D45-N47Δ-H6 used in the Example.

SEQ ID NO: 26 shows the amino acid sequence of one subunit ofHL-RQC-EcoExoIII-L2-D45-N47Δ-H6 used in the Example.

SEQ ID NO: 27 shows the polynucleotide sequence encodingHL-RQC-EcoExoI-Cter-{SG}8-H6 used in the Example.

SEQ ID NO: 28 shows the amino acid sequence of one subunit ofHL-RQC-EcoExoI-Cter-{SG}8-H6 used in the Example.

SEQ ID NO: 29 shows the polynucleotide sequence encodingHL-RQC-EcoExoI-Cter-DG{SG}8-H6 used in the Example.

SEQ ID NO: 30 shows the amino acid sequence of one subunit ofHL-RQC-EcoExoI-Cter-DG{SG}8-H6 used in the Example.

SEQ ID NOs: 31 and 32 show the oligonucleotide sequences used in theexonuclease assay of the Example.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “aconstruct” includes “constructs”, reference to “a transmembrane proteinpore” includes two or more such pores, reference to “a molecularadaptor” includes two or more such adaptors, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Constructs

The present invention provides constructs that are useful for sequencingnucleic acids. The constructs comprise a transmembrane protein poresubunit and a nucleic acid handling enzyme. The subunit is covalentlyattached to the enzyme. The constructs of the invention are useful toolsfor forming pores that are capable of sequencing nucleic acids bystochastic sensing. The constructs of the invention are particularlyuseful for generating transmembrane protein pores that can both handle atarget nucleic acid sequence and discriminate between the differentnucleotides in the target sequence. As described in more detail below,the enzyme handles a target nucleic acid in such a way that the pore canidentify nucleotides in the target sequence and thereby sequence thetarget sequence.

The subunit retains its ability to form a pore. The ability of aconstruct to form a pore can be assayed using any method known in theart. For instance, the construct may be inserted into a membrane alongwith other appropriate subunits and its ability to oligomerize to form apore may be determined. Methods are known in the art for insertingconstructs and subunits into membranes, such as lipid bilayers. Forexample, constructs and subunits may be suspended in a purified form ina solution containing a lipid bilayer such that it diffuses to the lipidbilayer and is inserted by binding to the lipid bilayer and assemblinginto a functional state. Alternatively, constructs and subunits may bedirectly inserted into the membrane using the “pick and place” methoddescribed in M. A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127,6502-6503 and International Application No. PCT/GB2006/001057 (publishedas WO 2006/100484). The ability of a construct to form a pore istypically assayed as described in the Examples.

The enzyme retains its ability to handle nucleic acids. This allows theconstruct to form a pore that may be used to sequence nucleic acids asdescribed below. The ability of a construct to handle nucleic acids canbe assayed using any method known in the art. For instance, construct orpores formed from the constructs can be tested for their ability tohandle specific sequences of nucleic acids. The ability of a constructor a pore to handle nucleic acids is typically assayed as described inthe Examples.

A construct of the invention may form part of a pore. Alternatively, aconstruct may be isolated, substantially isolated, purified orsubstantially purified. A construct is isolated or purified if it iscompletely free of any other components, such as lipids or other poremonomers. A construct is substantially isolated if it is mixed withcarriers or diluents which will not interfere with its intended use. Forinstance, a construct is substantially isolated or substantiallypurified if it present in a form that comprises less than 10%, less than5%, less than 2% or less than 1% of other components, such as lipids orother pore monomers. A construct of the invention may be present in alipid bilayer.

Attachment

The subunit is covalently attached to the enzyme. The subunit may beattached to the enzyme at more than one, such as two or three, points.Attaching the subunit to the enzyme at more than one point can be usedto constrain the mobility of the enzyme. For instance, multipleattachments may be used to constrain the freedom of the enzyme to rotateor its ability to move away from the subunit.

The subunit may be in a monomeric form when it is attached to the enzyme(post expression modification). Alternatively, the subunit may be partof an oligomeric pore when it is attached to an enzyme (postoligomerisation modification).

The subunit can be covalently attached to the enzyme using any methodknown in the art. The subunit and enzyme may be produced separately andthen attached together. The two components may be attached in anyconfiguration. For instance, they may be attached via their terminal(i.e. amino or carboxy terminal) amino acids. Suitable configurationsinclude, but are not limited to, the amino terminus of the enzyme beingattached to the carboxy terminus of the subunit and vice versa.Alternatively, the two components may be attached via amino acids withintheir sequences. For instance, the enzyme may be attached to one or moreamino acids in a loop region of the subunit. In a preferred embodiment,terminal amino acids of the enzyme are attached to one or more aminoacids in the loop region of a subunit. Terminal amino acids and loopregions are discussed above.

In one preferred embodiment, the subunit is genetically fused to theenzyme. A subunit is genetically fused to an enzyme if the wholeconstruct is expressed from a single polynucleotide sequence. The codingsequences of the subunit and enzyme may be combined in any way to form asingle polynucleotide sequence encoding the construct.

The subunit and enzyme may be genetically fused in any configuration.The subunit and enzyme may be fused via their terminal amino acids. Forinstance, the amino terminus of the enzyme may be fused to the carboxyterminus of the subunit and vice versa. The amino acid sequence of theenzyme is preferably added in frame into the amino acid sequence of thesubunit. In other words, the enzyme is preferably inserted within thesequence of the subunit. In such embodiments, the subunit and enzyme aretypically attached at two points, i.e. via the amino and carboxyterminal amino acids of the enzyme. If the enzyme is inserted within thesequence of the subunit, it is preferred that the amino and carboxyterminal amino acids of the enzyme are in close proximity and are eachattached to adjacent amino acids in the sequence of the subunit orvariant thereof. In a preferred embodiment, the enzyme is inserted intoa loop region of the subunit.

In another preferred embodiment, the subunit is chemically fused to theenzyme. A subunit is chemically fused to an enzyme if the two parts arechemically attached, for instance via a linker molecule.

The subunit may be transiently attached to the enzyme by a hex-his tagor Ni-NTA. The subunit and enzyme may also be modified such that theytransiently attach to each other.

The construct retains the pore forming ability of the subunit. The poreforming ability of the subunit is typically provided by its α-helicesand β-strands. β-barrel pores comprise a barrel or channel that isformed from β-strands, whereas α-helix bundle pores comprise a barrel orchannel that is formed from α-helices. The α-helices and β-strands aretypically connected by loop regions. In order to avoid affecting thepore forming ability of the subunit, the enzyme is preferablygenetically fused to a loop region of the subunit or inserted into aloop region of the subunit. The loop regions of specific subunits arediscussed in more detail below.

Similarly, the construct retains the nucleic acid handling ability ofthe enzyme, which is also typically provided by its secondary structuralelements (α-helices and β-strands) and tertiary structural elements. Inorder to avoid affecting the nucleic acid handling ability of theenzyme, the enzyme is preferably genetically fused to the subunit orinserted into the subunit via residues or regions that does not affectits secondary or tertiary structure.

The subunit may be attached directly to the enzyme. The subunit ispreferably attached to the enzyme using one or more, such as two orthree, linkers. The one or more linkers may be designed to constrain themobility of the enzyme. The linkers may be attached to one or morereactive cysteine residues, reactive lysine residues or non-naturalamino acids in the subunit and/or enzyme. Suitable linkers arewell-known in the art. Suitable linkers include, but are not limited to,chemical crosslinkers and peptide linkers. Preferred linkers are aminoacid sequences (i.e. peptide linkers). The length, flexibility andhydrophilicity of the peptide linker are typically designed such that itdoes not to disturb the functions of the subunit and enzyme. Preferredflexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10or 16, serine and/or glycine amino acids. More preferred flexiblelinkers include (SG)₁, (SG)₂. (SG)₃, (SG)₄, (SG)₅ and (SG)₈ wherein S isserine and G is glycine. Preferred rigid linkers are stretches of 2 to30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigidlinkers include (P)₁₂ wherein P is proline.

Linkers may be attached to the subunit first and then the enzyme, theenzyme first and then the subunit or the enzyme and subunit at the sametime. When the linker is attached to the subunit, it may be a monomericsubunit, part of an oligomer of two or more monomers or part of completeoligomeric pore. It is preferred that the linker is reacted before anypurification step to remove any unbound linker.

A preferred method of attaching the subunit to the enzyme is viacysteine linkage. This can be mediated by a bi-functional chemicallinker or by a polypeptide linker with a terminal presented cysteineresidue. α-HL (SEQ ID NO: 2) lacks native cysteine residues so theintroduction of a cysteine into the sequence of SEQ ID NO: 2 enables thecontrolled covalent attachment of the enzyme to the subunit. Cysteinescan be introduced at various positions, such as position KS, T9 or N17of SEQ ID NO: 2 or at the carboxy terminus of SEQ ID NO: 2. The length,reactivity, specificity, rigidity and solubility of any bi-functionallinker may designed to ensure that the enzyme is positioned correctly inrelation to the subunit and the function of both the subunit and enzymeis retained. Suitable linkers include bismaleimide crosslinkers, such as1,4-bis(maleimido)butane (BMB) or bis(maleimido)hexane. One draw back ofbi-functional linkers is the requirement of the enzyme to contain nofurther surface accessible cysteine residues, as binding of thebi-functional linker to these cannot be controlled and may affectsubstrate binding or activity. If the enzyme does contain severalaccessible cysteine residues, modification of the enzyme may be requiredto remove them while ensuring the modifications do not affect thefolding or activity of the enzyme. In a preferred embodiment, a reactivecysteine is presented on a peptide linker that is genetically attachedto the enzyme. This means that additional modifications will notnecessarily be needed to remove other accessible cysteine residues fromthe enzyme. The reactivity of cysteine residues may be enhanced bymodification of the adjacent residues, for example on a peptide linker.For instance, the basic groups of flanking arginine, histidine or lysineresidues will change the pKa of the cysteines thiol group to that of themore reactive S group. The reactivity of cysteine residues may beprotected by thiol protective groups such as dTNB. These may be reactedwith one or more cysteine residues of the enzyme or subunit, either as amonomer or part of an oligomer, before a linker is attached.

Cross-linkage of subunits or enzymes to themselves may be prevented bykeeping the concentration of linker in a vast excess of the subunitand/or enzyme. Alternatively, a “lock and key” arrangement may be usedin which two linkers are used. Only one end of each linker may reacttogether to form a longer linker and the other ends of the linker eachreact with a different part of the construct (i.e. subunit or monomer).

The site of covalent attachment is selected such that, when theconstruct is used to form a pore, the enzyme handles a target nucleicacid sequence in such a way that a proportion of the nucleotides in thetarget sequence interacts with the pore. Nucleotides are thendistinguished on the basis of the different ways in which they affectthe current flowing through the pore during the interaction.

There are a number of ways that pores can be used to sequence nucleicacid molecules. One way involves the use of an exonuclease enzyme, suchas a deoxyribonuclease. In this approach, the exonuclease enzyme is usedto sequentially detach the nucleotides from a target nucleic strand. Thenucleotides are then detected and discriminated by the pore in order oftheir release, thus reading the sequence of the original strand. Forsuch an embodiment, the exonuclease enzyme is preferably attached to thesubunit such that a proportion of the nucleotides released from thetarget nucleic acid is capable of entering and interacting with thebarrel or channel of a pore comprising the construct. The exonuclease ispreferably attached to the subunit at a site in close proximity to thepart of the subunit that forms the opening of the barrel of channel ofthe pore. The exonuclease enzyme is more preferably attached to thesubunit such that its nucleotide exit trajectory site is orientatedtowards the part of the subunit that forms part of the opening of thepore.

Another way of sequencing nucleic acids involves the use of an enzymethat pushes or pulls the target nucleic acid strand through the pore. Inthis approach, the ionic current fluctuates as a nucleotide in thetarget strand passes through the pore. The fluctuations in the currentare indicative of the sequence of the strand. For such an embodiment,the enzyme is preferably attached to the subunit such that it is capableof pushing or pulling the target nucleic acid through the barrel orchannel of a pore comprising the construct and does not interfere withthe flow of ionic current through the pore. The enzyme is preferablyattached to the subunit at a site in close proximity to the part of thesubunit that forms part of the opening of the barrel of channel of thepore. The enzyme is more preferably attached to the subunit such thatits active site is orientated towards the part of the subunit that formspart of the opening of the pore.

A third way of sequencing a nucleic acid strand is to detect thebi-products of a polymerase in close proximity to a pore detector. Inthis approach, nucleoside phosphates (nucleotides) are labelled so thata phosphate labelled species is released upon the addition of apolymerase to the nucleotide strand and the phosphate labelled speciesis detected by the pore. The phosphate species contains a specific labelfor each nucleotide. As nucleotides are sequentially added to thenucleic acid strand, the bi-products of the base addition are detected.The order that the phosphate labelled species are detected can be usedto determine the sequence of the nucleic acid strand.

The enzyme is preferably attached to a part of the subunit that formspart of the cis side of a pore comprising the construct. Inelectrophysiology, the cis side is the grounded side. If a hemolysinpore is inserted correctly into an electrophysiology apparatus, the Capregion is on the cis side. It is well known that, under a positivepotential, nucleotides will migrate from the cis to the trans side ofpores used for stochastic sensing. Positioning the enzyme at the cisside of a pore allows it to handle the target nucleic acid such that aproportion of the nucleotides in the sequence enters the barrel orchannel of the pore and interacts with it. Preferably, at least 20%, atleast 40%, at least 50%, at least 80% or at least 90% of the nucleotidesin the sequence enters the barrel or channel of the pore and interactswith it.

The site and method of covalent attachment is preferably selected suchthat mobility of the enzyme is constrained. This helps to ensure thatthe enzyme handles the target nucleic acid sequence in such a way that aproportion of the nucleotides in the target sequence interacts with thepore. For instance, constraining the ability of enzyme to move meansthat its active site can be permanently orientated towards the part ofthe subunit that forms part of the opening of the barrel of channel ofthe pore. The mobility of the enzyme may be constrained by increasingthe number of points at which the enzyme is attached to the subunitand/or the use of specific linkers.

Subunit

The constructs of the invention comprise a subunit from a transmembraneprotein pore. A transmembrane protein pore is a polypeptide or acollection of polypeptides that permits ions driven by an appliedpotential to flow from one side of a membrane. The pore preferablypermits nucleotides to flow from one side of a membrane to the otheralong the applied potential. The pore preferably allows a nucleic acid,such as DNA or RNA, to be pushed or pulled through the pore.

The subunit is part of a pore. The pore may be a monomer or an oligomer.The subunit preferably forms part of a pore made up of several repeatingsubunits, such as 6, 7 or 8 subunits. The subunit more preferably formspart of a heptameric pore. The subunit typically forms part of a barrelor channel through which the ions may flow. The subunits of the poretypically surround a central axis and contribute strands to atransmembrane β barrel or channel or a transmembrane α-helix bundle orchannel. When part of a construct of the invention, the subunit may be amonomer or part of an oligomeric pore.

The subunit typically forms part of a pore whose barrel or channelcomprises amino acids that facilitate interaction with nucleotides ornucleic acids. These amino acids are preferably located near theconstriction of the barrel or channel. The subunit typically comprisesone or more positively charged amino acids, such as arginine, lysine orhistidine. These amino acids typically facilitate the interactionbetween the pore and nucleotides or nucleic acids by interacting withthe phosphate groups in the nucleotides or nucleic acids or by π-cationinteraction with the bases in the nucleotides or nucleic acids. Thenucleotide detection can be facilitated with an adaptor.

Subunits for use in accordance with the invention can be derived fromβ-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrelor channel that is formed from β-strands. Suitable β-barrel poresinclude, but are not limited to, β-toxins, such as α-hemolysin andleukocidins, and outer membrane proteins/porins of bacteria, such asouter membrane porin F (OmpF), outer membrane porin G (OmpG), outermembrane phospholipase A and Neisseria autotransporter lipoprotein(NaIP). α-helix bundle pores comprise a barrel or channel that is formedfrom α-helices. Suitable α-helix bundle pores include, but are notlimited to, inner membrane proteins and α outer membrane proteins, suchas WZA.

The subunit is preferably derived from α-hemolysin (α-HL). The wild-typeα-HL pore is formed of seven identical monomers or subunits (i.e. it isheptameric). The sequence of one wild-type monomer or subunit ofα-hemolysin is shown in SEQ ID NO: 2. The subunit in the constructs ofthe invention preferably comprises the sequence shown in SEQ ID NO: 2 ora variant thereof. Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66,72, 92 to 97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to274, 287 to 290 and 294 of SEQ ID NO: 2 form loop regions. The enzyme ispreferably attached to one or more of amino acids 8, 9, 17, 18, 19, 44,45, 50 and 51 of SEQ ID NO: 2. The enzyme is more preferably insertedbetween amino acids, 18 and 19, 44 and 45 or 50 and 51 of SEQ ID NO: 2.

A variant of SEQ ID NO: 2 is a subunit that has an amino acid sequencewhich varies from that of SEQ ID NO: 2 and which retains its poreforming ability. The ability of the variant to form pores can be assayedas described above. The variant may include modifications thatfacilitate covalent attachment to or interaction with the nucleic acidhandling enzyme. The variant preferably comprises one or more reactivecysteine residues that facilitate attachment to the enzyme. Forinstance, the variant may include a cysteine at one or more of positions8, 9, 17, 18, 19, 44, 45, 50 and 51 and/or on the amino or carboxyterminus of SEQ ID NO: 2. Preferred variants comprise a substitution ofthe residue at position 8, 9 or 17 of SEQ ID NO: 2 with cysteine (K8C,T9C or N17C).

The variant may be modified to facilitate genetic fusion of the enzyme.For instance, one or more residues adjacent to the insertion site may bemodified, such as deleted, to facilitate insertion of the enzyme and/orlinkers. If the enzyme is inserted into loop 2 of SEQ ID NO: 2, one ormore of residues D45, K46, N47. H48, N49 and K50 of SEQ ID NO: 2 may bedeleted. A preferred construct containing such a deletion comprises thesequence shown in SEQ ID NO: 26 or a variant thereof.

The variant may also include modifications that facilitate anyinteraction with nucleotides or facilitate orientation of a molecularadaptor as discussed below. The variant may also contain modificationsthat facilitate covalent attachment of a molecular adaptor.

The subunit may be any of the variants of SEQ ID NO: 2 described in aco-pending International application claiming priority from USApplication No. 61/078,687 and being filed simultaneously with thisapplication [J A Kemp & Co Ref: N. 104403A; Oxford Nanolabs Ref: ONL IP004]. All the teachings of that application may be applied equally tothe present invention. In particular, the variant preferably has aglutamine at position 139 of SEQ ID NO: 2. The variant preferably has anarginine at position 113 of SEQ ID NO: 2. The variant preferably has acysteine at position 119, 121 or 135 of SEQ ID NO: 2. Any of thevariants of SEQ ID NO: 2 shown in SEQ ID NOs: 4, 6, 8, 10, 12 and 14 ofthe co-pending application may be used to form a construct of theinvention.

The subunit may be a naturally occurring variant which is expressed byan organism, for instance by a Staphylococcus bacterium. Variants alsoinclude non-naturally occurring variants produced by recombinanttechnology. Over the entire length of the amino acid sequence of SEQ IDNO: 2, a variant will preferably be at least 50% homologous to thatsequence based on amino acid identity. More preferably, the subunitpolypeptide may be at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 200 or more, for example 230,250, 270 or 280 or more, contiguous amino acids (“hard homology”).

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade, for example, according to Table 1 below.

TABLE 1 Conservative substitutions Amino acids in the same block in thesecond column and preferably in the same line in the third column may besubstituted for each other. NON-AROMATIC Non-polar G A P I L V Polar -uncharged C S T M N Q Polar - charged D E H K R AROMATIC H F W Y

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may fragments of SEQ ID NO: 2. Such fragments retain poreforming activity. Fragments may be at least 50, 100, 200 or 250 aminoacids in length. A fragment preferably comprises the pore forming domainof SEQ ID NO: 2. Fragments typically include residues 119, 121, 135, 113and 139 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 2 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a subunit or variant.

As discussed above, a variant of SEQ ID NO: 2 is a subunit that has anamino acid sequence which varies from that of SEQ ID NO: 2 and whichretains its ability to form a pore. A variant typically contains theregions of SEQ ID NO: 2 that are responsible for pore formation. Thepore forming ability of α-HL, which contains a 1-barrel, is provided byβ-strands in each subunit. A variant of SEQ ID NO: 2 typically comprisesthe regions in SEQ ID NO: 2 that form β-strands. The amino acids of SEQID NO: 2 that form β-strands are discussed above. One or moremodifications can be made to the regions of SEQ ID NO: 2 that formβ-strands as long as the resulting variant retains its ability to form apore. Specific modifications that can be made to the 1-strand regions ofSEQ ID NO: 2 are discussed above.

A variant of SEQ ID NO: 2 preferably includes one or more modifications,such as substitutions, additions or deletions, within its α-helicesand/or loop regions. Amino acids that form α-helices and loops arediscussed above.

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSP's containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two amino acidsequences would occur by chance. For example, a sequence is consideredsimilar to another sequence if the smallest sum probability incomparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The variant may be modified for example by the addition of histidine oraspartic acid residues to assist its identification or purification orby the addition of a signal sequence to promote their secretion from acell where the polypeptide does not naturally contain such a sequence.

The subunit may be labelled with a revealing label. The revealing labelmay be any suitable label which allows the pore to be detected. Suitablelabels include, but are not limited to, fluorescent molecules,radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

The subunit may be isolated from a pore producing organism, such asStaphylococcus aureus, or made synthetically or by recombinant means.For example, the subunit may be synthesized by in vitro translation andtranscription. The amino acid sequence of the subunit may be modified toinclude non-naturally occurring amino acids or to increase the stabilityof the subunit. When the subunit is produced by synthetic means, suchamino acids may be introduced during production. The subunit may also bealtered following either synthetic or recombinant production.

The subunit may also be produced using D-amino acids. For instance, thepores may comprise a mixture of L-amino acids and D-amino acids. This isconventional in the art for producing such proteins or peptides.

The subunit may also contain other non-specific chemical modificationsas long as they do not interfere with its ability to form a pore. Anumber of non-specific side chain modifications are known in the art andmay be made to the side chains of the pores. Such modifications include,for example, reductive alkylation of amino acids by reaction with analdehyde followed by reduction with NaBH₄, amidination withmethylacetimidate or acylation with acetic anhydride. The modificationsto the subunit can be made after expression of the subunit or constructor after the subunit has been used to form a pore.

The subunit can be produced using standard methods known in the art.Polynucleotide sequences encoding a subunit may be isolated andreplicated using standard methods in the art. Such sequences arediscussed in more detail below. Polynucleotide sequences encoding asubunit may be expressed in a bacterial host cell using standardtechniques in the art. The subunit may be produced in a cell by in situexpression of the polypeptide from a recombinant expression vector. Theexpression vector optionally carries an inducible promoter to controlthe expression of the polypeptide.

A subunit may be produced in large scale following purification by anyprotein liquid chromatography system from pore producing organisms orafter recombinant expression as described below. Typical protein liquidchromatography systems include FPLC, AKTA systems, the Bio-Cad system,the Bio-Rad BioLogic system and the Gilson HPLC system.

Nucleic Acid Handling Enzyme

The constructs of the invention comprise a nucleic acid handling enzyme.A nucleic acid handling enzyme is a polypeptide that is capable ofinteracting with and modifying at least one property of a nucleic acid.The enzyme may modify the nucleic acid by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The enzyme may modify the nucleic acid by orienting itor moving it to a specific position.

A nucleic acid is a macromolecule comprising two or more nucleotides.The nucleic acid handled by the enzyme may comprise any combination ofany nucleotides. The nucleotides can be naturally occurring orartificial. A nucleotide typically contains a nucleobase, a sugar and atleast one phosphate group. The nucleobase is typically heterocyclic.Nucleobases include, but are not limited to, purines and pyrimidines andmore specifically adenine, guanine, thymine, uracil and cytosine. Thesugar is typically a pentose sugar. Nucleotide sugars include, but arenot limited to, ribose and deoxyribose. The nucleotide is typically aribonucleotide or deoxyribonucleotide. The nucleotide typically containsa monophosphate, diphosphate or triphosphate. Phosphates may be attachedon the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP,dTMP, dGMP or dCMP.

The nucleic acid handled by the enzyme is preferably double stranded,such as DNA. The nucleic acid handled by the enzyme may be singlestranded, such as cDNA or RNA. Enzymes that handle single strandednucleic acids may be used to sequence double stranded DNA as long as thedouble stranded DNA is chemically or thermally dissociated into a singlestrand before it is handled by the enzyme.

It is preferred that the tertiary structure of the nucleic acid handlingenzyme is known. Knowledge of the three dimensional structure of theenzyme allows modifications to be made to the enzyme to facilitate itsfunction in the construct or pore of the invention.

The enzyme may be any size and have any structure. For instance, theenzyme may be an oligomer, such as a dimer or trimer. The enzyme ispreferably a small, gloubular polypeptide formed from one monomer. Suchenzymes are easy to handle and are less likely to interfere with thepore forming ability of the subunit, particularly if fused to orinserted into the sequence of the subunit.

The amino and carboxy termini of the enzyme are preferably in closeproximity. The amino and carboxy termini of the enzyme are morepreferably presented on same face of the enzyme. Such embodimentsfacilitate insertion of the enzyme into the sequence of the subunit. Forinstance, if the amino and carboxy termini of the enzyme are in closeproximity, each can be attached by genetic fusion to adjacent aminoacids in the sequence of the subunit.

It is also preferred that the location and function of the active siteof the enzyme is known. This prevents modifications being made to theactive site that abolish the activity of the enzyme. It also allows theenzyme to be attached to the subunit so that the enzyme handles thetarget nucleic acid sequence in such a way that a proportion of thenucleotides in the target sequence interacts with the pore. It isbeneficial to position the active site of the enzyme as close aspossible to the part of the subunit that forms part of the opening ofthe barrel of channel of the pore, without the enzyme itself presentinga block to the flow of current. Knowledge of the way in which an enzymemay orient nucleic acids also allows an effective construct to bedesigned.

As discussed in more detail below, it may be necessary to purify theconstruct of the invention. It is preferred that the enzyme is capableof withstanding the conditions used to purify the construct.

The constructs of the invention are useful for forming pores. Such poresmay be used to sequence nucleic acids. In order that most of thenucleotides in the target nucleic acid are correctly identified bystochastic sensing, the enzyme must handle the nucleic acid in a bufferbackground which is compatible with discrimination of the nucleotides.The enzyme preferably has at least residual activity in a saltconcentration well above the normal physiological level, such as from100 mM to 500 mM. The enzyme is more preferably modified to increase itsactivity at high salt concentrations. The enzyme may also be modified toimprove its processivity, stability and shelf life.

Suitable modifications can be determined from the characterisation ofnucleic acid handling enzymes from extremophiles such as halophilic,moderately halophilic bacteria, thermophilic and moderately thermophilicorganisms, as well as directed evolution approaches to altering the salttolerance, stability and temperature dependence of mesophilic orthermophilic exonucleases.

The enzyme also preferably retains at least partial activity at roomtemperature. This allows pores formed from the construct to sequencenucleic acids at room temperature.

The nucleic acid handling enzyme is preferably a nucleolytic enzyme. Thenucleic acid handling enzyme is more preferably member of any of theEnzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Thenucleic acid handling enzyme is more preferably any one of the followingenzymes:

-   -   3.1.11.—Exodeoxyribonucleases producing 5′-phosphomonoesters.        -   3.1.11.1 Exodeoxyribonuclease I.        -   3.1.11.2 Exodeoxyribonuclease III.        -   3.1.11.3 Exodeoxyribonuclease (lambda-induced).        -   3.1.11.4 Exodeoxyribonuclease (phage SP3-induced).        -   3.1.11.5 Exodeoxyribonuclease V.        -   3.1.11.6 Exodeoxyribonuclease VII.    -   3.1.13.—Exoribonucleases producing 5′-phosphomonoesters.        -   3.1.13.1 Exoribonuclease II.        -   3.1.13.2 Exoribonuclease H.        -   3.1.13.3 Oligonucleotidase.        -   3.1.13.4 Poly(A)-specific ribonuclease.        -   3.1.13.5 Ribonuclease D.    -   3.1.14.—Exoribonucleases producing 3′-phosphomonoesters.        -   3.1.14.1 Yeast ribonuclease.    -   3.1.15.—Exonucleases active with either ribo- or        deoxyribonucleic acid producing 5′ phosphomonoesters        -   3.1.15.1 Venom exonuclease.    -   3.1.16.—Exonucleases active with either ribo- or        deoxyribonucleic acid producing 3′ phosphomonoesters        -   3.1.16.1 Spleen exonuclease.    -   3.1.21.—Endodeoxyribonucleases producing 5′-phosphomonoesters.        -   3.1.21.1 Deoxyribonuclease 1.        -   3.1.21.2 Deoxyribonuclease IV (phage-T(4)-induced).        -   3.1.21.3 Type I site-specific deoxyribonuclease.        -   3.1.21.4 Type II site-specific deoxyribonuclease.        -   3.1.21.5 Type III site-specific deoxyribonuclease.        -   3.1.21.6 CC-preferring endodeoxyribonuclease.        -   3.1.21.7 Deoxyribonuclease V.    -   3.1.22.—Endodeoxyribonucleases producing other than        5′-phosphomonoesters.        -   3.1.22.1 Deoxyribonuclease II.        -   3.1.22.2 Aspergillus deoxyribonuclease K(1).        -   3.1.22.3 Transferred entry: 3.1.21.7.        -   3.1.22.4 Crossover junction endodeoxyribonuclease.        -   3.1.22.5 Deoxyribonuclease X.    -   3.1.25.—Site-specific endodeoxyribonucleases specific for        altered bases.        -   3.1.25.1 Deoxyribonuclease (pyrimidine dimer).        -   3.1.25.2 Transferred entry: 4.2.99.18.    -   3.1.26.—Endoribonucleases producing 5′-phosphomonoesters.        -   3.1.26.1 Physarum polycephalum ribonuclease.        -   3.1.26.2 Ribonuclease alpha.        -   3.1.26.3 Ribonuclease III.        -   3.1.26.4 Ribonuclease H.        -   3.1.26.5 Ribonuclease P.        -   3.1.26.6 Ribonuclease IV.        -   3.1.26.7 Ribonuclease P4.        -   3.1.26.8 Ribonuclease M5.        -   3.1.26.9 Ribonuclease (poly-(U)-specific).        -   3.1.26.10 Ribonuclease IX.        -   3.1.26.11 Ribonuclease Z.    -   3.1.27.—Endoribonucleases producing other than        5′-phosphomonoesters.        -   3.1.27.1 Ribonuclease T(2).        -   3.1.27.2 Bacillus subtilis ribonuclease.        -   3.1.27.3 Ribonuclease T(1).        -   3.1.27.4 Ribonuclease U(2).        -   3.1.27.5 Pancreatic ribonuclease.        -   3.1.27.6 Enterobacter ribonuclease.        -   3.1.27.7 Ribonuclease F.        -   3.1.27.8 Ribonuclease V.        -   3.1.27.9 tRNA-intron endonuclease.        -   3.1.27.10 rRNA endonuclease.    -   3.1.30.—Endoribonucleases active with either ribo- or        deoxyribonucleic producing 5′ phospomonoesters        -   3.1.30.1 Aspergillus nuclease S(1).        -   3.1.30.2 Serratia marcescens nuclease.    -   3.1.31.—Endoribonucleases active with either ribo- or        deoxyribonucleic producing 3′ phosphomonoesters        -   3.1.31.1 Micrococcal nuclease.

The enzyme is most preferably an exonuclease, such as adeoxyribonuclease, which cleave nucleic acids to form individualnucleotides. The advantages of exodeoxyribonucleases are that they areactive on both single stranded and double stranded DNA and hydrolysebases either in either the 5′-3′ or 3′-5′ direction.

An individual nucleotide is a single nucleotide. An individualnucleotide is one which is not bound to another nucleotide or nucleicacid by a nucleotide bond. A nucleotide bond involves one of thephosphate groups of a nucleotide being bound to the sugar group ofanother nucleotide. An individual nucleotide is typically one which isnot bound by a nucleotide bond to another nucleic acid sequence of atleast 5, at least 10, at least 20, at least 50, at least 100, at least200, at least 500, at least 1000 or at least 5000 nucleotides.

Preferred enzymes for use in the method include exonuclease III enzymefrom E. coli (SEQ ID NO: 10), exonuclease I from E. coli (SEQ ID NO:12), RecJ from T. thermophilus (SEQ ID NO: 14) and bacteriophage lambdaexonuclease (SEQ ID NO: 16) and variants thereof. The exonuclease enzymepreferably comprises any of the sequences shown in SEQ ID NOs: 10, 12,14 and 16 or a variant thereof. Three identical subunits of SEQ ID NO:16 interact to form a trimer exonuclease. A variant of SEQ ID NO: 10,12, 14 or 16 is an enzyme that has an amino acid sequence which variesfrom that of SEQ ID NO: 10, 12, 14 or 16 and which retains nucleic acidhandling ability. The enzyme may include modifications that facilitatehandling of the nucleic acid and/or facilitate its activity at high saltconcentrations and/or room temperature. The enzyme may includemodifications that facilitate covalent attachment to or its interactionwith the subunit. As discussed above, accessible cysteines may beremoved from the enzyme to avoid non-specific reactions with a linker.Alternatively, one or more reactive cysteines may be introduced into theenzyme, for instance as part of a genetically-fused peptide linker, tofacilitate attachment to the subunit.

Variants may differ from SEQ ID NO: 10, 12, 14 and 16 to the same extentas variants of SEQ ID NO: 2 differ from SEQ ID NO: 2 as discussed above.

A variant of SEQ ID NO: 10, 12, 14 or 16 retains its nucleic acidhandling activity. A variant typically contains the regions of SEQ IDNO: 10, 12, 14 or 16 that are responsible for nucleic acid handlingactivity. The catalytic domains of SEQ ID NOs: 10, 12, 14 and 16 arediscussed above. A variant of SEQ ID NO: 10, 12, 14 or 16 preferablycomprises the relevant catalytic domain. A variant SEQ ID NO: 10, 12, 14or 16 typically includes one or more modifications, such assubstitutions, additions or deletions, outside the relevant catalyticdomain.

Preferred enzymes that are capable of pushing or pulling the targetnucleic acid sequence through the pore include polymerases,exonucleases, helicases and topoisomerases, such as gyrases. Thepolymerase is preferably a member of any of the Enzyme Classification(EC) groups 2.7.7.6, 2.7.7.7, 2.7.7.19, 2.7.7.48 and 2.7.7.49. Thepolymerase is preferably a DNA-dependent DNA polymerase, anRNA-dependent DNA polymerase, a DNA-dependent RNA polymerase or anRNA-dependent RNA polymerase. The helicase is preferably a member of anyof the Enzyme Classification (EC) groups 3.6.1.- and 2.7.7.-. Thehelicase is preferably an ATP-dependent DNA helicase (EC group 3.6.1.8),an ATP-dependent RNA helicase (EC group 3.6.1.8) or an ATP-independentRNA helicase. The topoisomerase is preferably a member of any of theEnzyme Classification (EC) groups 5.99.1.2 and 5.99.1.3.

The enzyme may be labelled with a revealing label. The revealing labelmay be any of those described above.

The enzyme may be isolated from an enzyme-producing organism, such as E.coli, T. thermophilus or bacteriophage, or made synthetically or byrecombinant means. For example, the enzyme may be synthesized by invitro translation and transcription as described above and below. Theenzyme may be produced in large scale following purification asdescribed above.

Preferred Constructs

Preferred constructs of the invention comprise the sequence shown in anyone of SEQ ID NOs: 18, 20, 22, 24, 26, 28 and 30 or a variant thereof.Variants of SEQ ID NO: 18, 20, 22, 24, 26, 28 or 30 must retain theirpore forming ability and nucleic acid handling ability. Variants maydiffer from SEQ ID NOs: 18, 20, 22, 24, 26, 28 and 30 to the same extentand in the same way as discussed above for variants of SEQ ID NO: 2 andvariants of SEQ ID NO: 10, 12, 14 or 16.

Polynucleotide Sequences

The present invention also provides polynucleotide sequences whichencode a construct in which the enzyme is genetically fused to thesubunit or is inserted into the sequence of the subunit. It isstraightforward to generate such polynucleotide sequences using standardtechniques. A polynucleotide sequence encoding the enzyme is eitherfused to or inserted into a polynucleotide sequence encoding thesubunit. The fusion or insertion is typically in frame. If apolynucleotide sequence encoding the enzyme is inserted into apolynucleotide sequence encoding the subunit, the sequence encoding theenzyme is typically flanked at both ends by restriction endonucleasesites, such as those recognized by BspE1. It may also be flanked at bothends by polynucleotide sequences encoding linkers, such as 5 to 10codons each encoding serine or glycine.

The polynucleotide sequence preferably encodes a construct comprisingSEQ ID NO: 10, 12, 14 or 16 or a variant thereof genetically fused to orinserted into SEQ ID NO: 2 or a variant thereof. The variants of SEQ IDNO: 2, 10, 12, 14 or 16 may be any of those discussed above. SEQ ID NO:10, 12, 14 or 16 or a variant thereof may be genetically fused to orinserted into SEQ ID NO: 2 or a variant thereof as described above.

The polynucleotide sequence preferably comprises SEQ ID NO: 9, 11, 13 or15 or a variant thereof genetically fused to or inserted into SEQ ID NO:1 or a variant thereof. SEQ ID NO: 9, 11, 13 or 15 or a variant thereofis preferably inserted into SEQ ID NO: 1 or a variant thereof betweennucleotides 2765 and 2766, 2843 and 2844 or 2861 and 2862 of SEQ IDNO: 1. The polynucleotide sequence more preferably comprises thesequence shown in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 29 or a variantthereof.

Variants of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29are sequences that are at least 50%, 60%, 70%, 80%, 90% or 95%homologous based on nucleotide identity to sequence of SEQ ID NO: 1, 9,11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 over the entire sequence. Theremay be at least 80%, for example at least 85%, 90% or 95% nucleotideidentity over a stretch of 600 or more, for example 700, 750, 850 or 900or more, contiguous nucleotides (“hard homogly”). Homology may becalculated as described above. The polynucleotide sequence may comprisea sequence that differs from SEQ ID NO: 1, 9, 11, 13, 15, 17, 19, 21,23, 25, 27 or 29 on the basis of the degeneracy of the genetic code.

Polynucleotide sequences may be isolated and replicated using standardmethods in the art. Chromosomal DNA may be extracted from a poreproducing organism, such as Staphylococcus aureus, and/or an enzymeproducing organism, such as E. coli, T. thermophilus or bacteriophage.The gene encoding the subunit and enzyme may be amplified using PCRinvolving specific primers. The amplified sequences may then beincorporated into a recombinant replicable vector such as a cloningvector. The vector may be used to replicate the polynucleotide in acompatible host cell. Thus polynucleotide sequences encoding a subunitand/or enzyme may be made by introducing a polynucleotide encoding asubunit and/or enzyme into a replicable vector, introducing the vectorinto a compatible host cell, and growing the host cell under conditionswhich bring about replication of the vector. The vector may be recoveredfrom the host cell. Suitable host cells for cloning of polynucleotidesare known in the art and described in more detail below.

The polynucleotide sequence may be cloned into suitable expressionvector. In an expression vector, the polynucleotide sequence encoding aconstruct is typically operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by thehost cell. Such expression vectors can be used to express a construct.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. Multiple copies of the same or different polynucleotide maybe introduced into the vector.

The expression vector may then be introduced into a suitable host cell.Thus, a construct can be produced by inserting a polynucleotide sequenceencoding a construct into an expression vector, introducing the vectorinto a compatible bacterial host cell, and growing the host cell underconditions which bring about expression of the polynucleotide sequence.The recombinantly-expressed construct may self-assemble into a pore inthe host cell membrane. Alternatively, the recombinant constructproduced in this manner may be isolated from the host cell and insertedinto another membrane. When producing an oligomeric pore comprising aconstruct of the invention and at least one different subunit, theconstruct and different subunits may be expressed separately indifferent host cells as described above, removed from the host cells andassembled into a pore in a separate membrane, such as a rabbit cellmembrane.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide sequence and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. A T7, trc, lac,ara or λ_(L) promoter is typically used.

The host cell typically expresses the construct at a high level. Hostcells transformed with a polynucleotide sequence encoding a constructwill be chosen to be compatible with the expression vector used totransform the cell. The host cell is typically bacterial and preferablyE. coli. Any cell with a λ DE3 lysogen, for example C41 (DE3), BL21(DE3), JM109 (DE3), B834 (DE3), TUNER. Origami and Origami B, canexpress a vector comprising the T7 promoter.

Modified Pores

The present invention also provides modified pores for use in sequencingnucleic acids. The pores comprise at least one construct of theinvention. The pores may comprise more than one, such as 2, 3 or 4,constructs of the invention.

A pore of the invention may be isolated, substantially isolated,purified or substantially purified. A pore of the invention is isolatedor purified if it is completely free of any other components, such aslipids or other pores. A pore is substantially isolated if it is mixedwith carriers or diluents which will not interfere with its intendeduse. For instance, a pore is substantially isolated or substantiallypurified if it present in a form that comprises less than 10%, less than5%, less than 2% or less than 1% of other components, such as lipids orother pores. Alternatively, a pore of the invention may be present in alipid bilayer or in a surfactant micelle.

The enzyme attached to the construct handles a target nucleic acidsequence in such a way that a proportion of the nucleotide in the targetsequence interacts with the pore, preferably the barrel or channel ofthe pore. Nucleotides are then distinguished on the basis of thedifferent ways in which they affect the current flowing through the poreduring the interaction.

The fixed nature of the enzyme means that a target nucleic acid sequenceis handled by the pore in a specific manner. For instance, eachnucleotide may be digested from one of the target sequence in aprocessive manner or the target sequence may be pushed or pulled throughthe pore. This ensures that a proportion of the nucleotides in thetarget nucleic acid sequence interacts with the pore and is identified.The lack of any interruption in the signal is important when sequencingnucleic acids. In addition, the fixed nature of the enzyme and the poremeans they can be stored together, thereby allowing the production of aready-to-use sensor.

In a preferred embodiment, an exonuclease enzyme, such as adeoxyribonuclease, is attached to the pore such that a proportion of thenucleotides is released from the target nucleic acid and interacts withthe barrel or channel of the pore. In another preferred embodiment, anenzyme that is capable of pushing or pulling the target nucleic acidsequence through the pore is attached to the pore such that the targetnucleic acid sequence is pushed or pulled through the barrel or channelof the pore and a proportion of the nucleotides in the target sequenceinteracts with the barrel or channel. In this embodiment, thenucleotides may interact with the pore in blocks or groups of more thanone, such as 2, 3 or 4. Suitable enzymes include, but are not limitedto, polymerases, exonucleases, helicases and topoisomerases, such asgyrases. In each embodiment, the enzyme is preferably attached to thepore at a site in close proximity to the opening of the barrel ofchannel of the pore. The enzyme is more preferably attached to the poresuch that its active site is orientated towards the opening of thebarrel of channel of the pore. This means that a proportion of thenucleotides of the target nucleic acid sequence is fed in the barrel orchannel. The enzyme is preferably attached to the cis side of the pore.

The modified pore may be based on any of the transmembrane protein poresdiscussed above, including the β-barrel pores and α-helix bundle pores.

For constructs comprising the sequence shown in SEQ ID NO: 2 or avariant thereof, the pore typically comprises an appropriate number ofadditional subunits comprising the sequence shown in SEQ ID NO: 2 or avariant thereof. A preferred pore of the invention comprises oneconstruct comprising the sequence shown in SEQ ID NO: 2 or a variantthereof and six subunits comprising the sequence shown in SEQ ID NO: 2or a variant thereof. The pore may comprise one or more subunitscomprising the sequence shown in SEQ ID NO: 4 or a variant thereof. SEQID NO: 4 shows the sequence of SEQ ID NO: 2 except that it has anarginine at position 113 (M113R) and a glutamine at position 139(N139Q). A variant of SEQ ID NO: 4 may differ from SEQ ID NO: 4 in thesame way and to the same extent as discussed for SEQ ID NO: 2 above. Apreferred pore of the invention comprises one construct comprising thesequence shown in SEQ ID NO: 2 or a variant thereof and six subunitscomprising the sequence shown in SEQ ID NO: 4 or a variant thereof.

The pores may comprise a molecular adaptor that facilitates theinteraction between the pore and the nucleotides or the target nucleicacid sequence. The presence of the adaptor improves the host-guestchemistry of the pore and nucleotides released from or present in thetarget nucleic acid sequence. The principles of host-guest chemistry arewell-known in the art. The adaptor has an effect on the physical orchemical properties of the pore that improves its interaction withnucleotides. The adaptor typically alters the charge of the barrel orchannel of the pore or specifically interacts with or binds tonucleotides thereby facilitating their interaction with the pore.

The adaptor mediates the interaction between nucleotides released fromor present in the target nucleic acid sequence and the pore. Thenucleotides preferably reversibly bind to the pore via or in conjunctionwith the adaptor. The nucleotides most preferably reversibly bind to thepore via or in conjunction with the adaptor as they pass through thepore across the membrane. The nucleotides can also reversibly bind tothe barrel or channel of the pore via or in conjunction with the adaptoras they pass through the pore across the membrane. The adaptorpreferably constricts the barrel or channel so that it may interact withthe nucleotides.

The adaptor is typically cyclic. The adaptor preferably has the samesymmetry as the pore. An adaptor having seven-fold symmetry is typicallyused if the pore is heptameric (e.g. has seven subunits around a centralaxis that contribute 14 strands to a transmembrane β barrel). Likewise,an adaptor having six-fold symmetry is typically used if the pore ishexameric (e.g. has six subunits around a central axis that contribute12 strands to a transmembrane β barrel, or is a 12-stranded β barrel).Any adaptor that that facilitates the interaction between the pore andthe nucleotide can be used. Suitable adaptors include, but are notlimited to, cyclodextrins, cyclic peptides and cucurbiturils. Theadaptor is preferably a cyclodextrin or a derivative thereof. Theadaptor is more preferably heptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). Table 2 belowshows preferred combinations of pores and adaptors.

TABLE 2 Suitable combinations of pores and adaptors Number of strands inthe transmembrane Pore β-barrel Adaptor Leukocidin 16 γ-cyclodextrin(γ-CD) OmpF 16 γ-cyclodextrin (γ-CD) α-hemolysin (or a 14 β-cyclodextrin(β-CD) variant thereof 6-monodeoxy-6- discussed above)monoamino-β-cyclodextrin (am₁β-CD) heptakis-6-amino-β- cyclodextrin(am₇-β-CD) heptakis-(6-deoxy-6- guanidino)-cyclodextrin (gu₇-β-CD) OmpG14 β-cyclodextrin (β-CD) 6-monodeoxy-6- monoamino-β-cyclodextrin(am₁β-CD) heptakis-6-amino-β- cyclodextrin (am₇-β-CD)heptakis-(6-deoxy-6- guanidino)-cyclodextrin (gu₇-β-CD) NalP 12α-cyclodextrin (α-CD) OMPLA 12 α-cyclodextrin (α-CD)

The adaptor is preferably covalently attached to the pore. The adaptorcan be covalently attached to the pore using any method known in theart. The adaptor may be attached directly to the pore. The adaptor ispreferably attached to the pore using a bifunctional crosslinker.Suitable crosslinkers are well-known in the art. Preferred crosslinkersinclude 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate,2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate and2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate. The mostpreferred crosslinker is succinimidyl 3-(2-pyridyldithio)propionate(SPDP). Typically, the adaptor is covalently attached to thebifunctional crosslinker before the adaptor/crosslinker complex iscovalently attached to the pore but it is also possible to covalentlyattach the bifunctional crosslinker to the pore before the bifunctionalcrosslinker/pore complex is attached to the adaptor.

The site of covalent attachment is selected such that the adaptorfacilitates interaction of nucleotides released from or present in thetarget nucleic acid sequence with the pore and thereby allows detectionof nucleotides. This can be done as explained in the co-pendingInternational application claiming priority from U.S. Application No.61/078,687 and being filed simultaneously with this application [J AKemp & Co Ref: N. 104403A; Oxford Nanolabs Ref: ONL IP 004].

For pores based on α-HL, the correct orientation of the adaptor withinthe barrel or channel of the pore and the covalent attachment of adaptorto the pore can be facilitated as described in the co-pendingInternational application claiming priority from U.S. Application No.61/078,687 and being filed simultaneously with this application [J AKemp & Co Ref: N.104403A; Oxford Nanolabs Ref: ONL IP 004]. Any of thespecific modifications to SEQ ID NO: 2 disclosed in the co-pendingapplication are equally applicable to the pores of this invention. Inparticular, every subunit of the pore, including the construct(s),preferably has a glutamine at position 139 of SEQ ID NO: 2. One or moreof the subunits of the pore, including the construct(s), may have anarginine at position 113 of SEQ ID NO: 2. One or more of the subunits ofthe pore, including the construct(s), may have a cysteine at position119, 121 or 135 of SEQ ID NO: 2. Any of the variants of SEQ ID NO: 2shown in SEQ ID NOs: 4, 6, 8, 10, 12 and 14 of the co-pendingapplication may be used to form a modified pore of the invention.

Preferred modified pores of the invention comprise:

(a) a construct comprising the sequence shown in SEQ ID NO: 18, 20, 22,24, 26, 28 or 30 or a variant thereof and six subunits of α-HLM113R/N139Q shown in SEQ ID NO: 4;

(b) a construct of the invention comprising the sequence shown in SEQ IDNO: 2 or a variant thereof, five subunits of α-HL M113R/N139Q shown inSEQ ID NO: 4 or a variant thereof and one subunit of α-HLM113R/N139Q/G119C-D8 shown in SEQ ID NO: 10 of the co-pendingapplication;

(c) a a construct of the invention comprising the sequence shown in SEQID NO: 2 or a variant thereof, five subunits of α-HL M113R/N139Q shownin SEQ ID NO: 4 or a variant thereof and one subunit of α-HLM113R/N139Q/N121C-D8 shown in SEQ ID NO: 12 of the co-pendingapplication; or

(d) a construct of the invention comprising the sequence shown in SEQ IDNO: 2 or a variant thereof, five subunits of α-HL M113R/N139Q shown inSEQ ID NO: 4 or a variant thereof and and one subunit of α-HLM113R/N139Q/L135C-D8 shown in SEQ ID NO: 14 of the co-pendingapplication.

Methods of Producing Constructs of the Invention

The invention also provides methods of producing a construct of theinvention. The methods comprise covalently attaching a nucleic acidhandling enzyme to a transmembrane protein pore subunit. Any of thesubunits and enzymes discussed above can be used in the methods. Thesite of and method of covalent attachment are selected as discussedabove.

The methods also comprise determining whether or not the construct iscapable of forming a pore and handling nucleic acids. Assays for doingthis are described above. If a pore can be formed and nucleic acids canbe handled, the subunit and enzyme have been attached correctly and aconstruct of the invention has been produced. If a pore cannot be formedor nucleic acids cannot be handled, a construct of the invention has notbeen produced.

Methods of Producing Modified Pores

The present invention also provides methods of producing modified poresof the invention. The modified pore may be formed by allowing at leastone construct of the invention to form a pore with other suitablesubunits or by covalently attaching an enzyme to a subunit in anoligomeric pore. Any of the constructs, subunits, enzymes or poresdiscussed above can be used in the methods. The site of and method ofcovalent attachment are selected as discussed above.

The methods also comprise determining whether or not the pore is capableof handling nucleic acids and detecting nucleotides. The pore may beassessed for its ability to detect individual nucleotides or shortchains of nucleotides, such as di- or trinucleotides. Assays for doingthis are described above and below. If the pore is capable of handlingnucleic acids and detecting nucleotides, the subunit and enzyme havebeen attached correctly and a pore of the invention has been produced.If a pore cannot be handle nucleic acids and detect nucleotides, a poreof the invention has not been produced.

In a preferred embodiment, a heteroheptamer of seven subunits comprisingthe sequence shown in SEQ ID NO: 2 or a variant thereof and containingone cysteine in an appropriate place is reacted with a bifunctionalcross-linker. The pore may be reacted with the linker before or after ithas been purified, typically by SDS PAGE. The pore/linker construct isthen reacted with an enzyme containing at least one reactive cysteine,for instance on a genetically-fused peptide linker. After the couplingreaction, the modified pore of the invention is removed from anyunreacted enzyme or pore/linker construct.

Method of Purifying Pores

The present invention also provides methods of purifying modified poresof the invention. The methods allow the purification of pores comprisingat least one construct of the invention. The methods do not involve theuse of anionic surfactants, such as sodium dodecyl sulphate (SDS), andtherefore avoid any detrimental effects on the enzyme part of theconstruct. The methods are particularly good for purifying porescomprising a construct of the invention in which the subunit and enzymehave been genetically fused.

The methods involve providing at least one construct of the inventionand any remaining subunits required to form a pore of the invention. Anyof the constructs and subunits discussed above can be used. Theconstruct(s) and remaining subunits are inserted into synthetic lipidvesicles and allowed to oligomerise. Methods for inserting theconstruct(s) and remaining subunits into synthetic vesicles are wellknown in the art.

The synthetic vesicles should have similar properties to rabbit cellmembranes, but should lack the rabbit cell membrane proteins. Thevesicles may comprise any components and are typically made of a blendof lipids. Suitable lipids are well-known in the art. The syntheticvesicles preferably comprise 30% cholesterol, 30% phosphatidylcholine(PC), 20% phosphatidylethanolamine (PE), 10% sphingomyelin (SM) and 10%phosphatidylserine (PS).

The vesicles are then contacting with a non-ionic surfactant or a blendof non-ionic surfactants. The non-ionic surfactant is preferably anOctyl Glucoside (OG) or DoDecyl Maltoside (DDM) detergent. Theoligomerised pores are then purified, for example by using affinitypurification based on his-tag or Ni-NTA.

Methods of Sequencing Nucleic Acids

The present invention also provides methods of sequencing a targetnucleic acid sequence. In one embodiment, the method comprises (a)contacting the target sequence with a pore of the invention, whichcomprises an exonuclease, such that the exonuclease digests anindividual nucleotide from one end of the target sequence; (b)contacting the nucleotide with the pore so that the nucleotide interactswith the adaptor, (c) measuring the current passing through the poreduring the interaction and thereby determining the identity of thenucleotide; and (d) repeating steps (a) to (c) at the same end of thetarget sequence and thereby determining the sequence of the targetsequence. Hence, the method involves stochastic sensing of a proportionof the nucleotides in a target nucleic acid sequence in a successivemanner in order to sequence the target sequence. Individual nucleotidesare described above.

In another embodiment, the method comprises (a) contacting the targetsequence with a pore of the invention so that the target sequence ispushed or pulled through the pore and a proportion of the nucleotides inthe target sequence interacts with the pore and (b) measuring thecurrent passing through the pore during each interaction and therebydetermining the sequence of the target sequence. Hence, the methodinvolves stochastic sensing of a proportion of the nucleotides in atarget nucleic acid sequence as the nucleotides pass through the barrelor channel in a successive manner in order to sequence the targetsequence.

Pores comprising a construct of the invention are particularly suited tothese methods. In order to effectively sequence the nucleic acid, it isimportant to ensure that a proportion of the nucleotides in the nucleicacid is identified in a successive manner. The fixed nature of theenzyme means that a proportion of the nucleotides in the target sequenceaffects the current flowing through the pore.

The whole or only part of the target nucleic acid sequence may besequenced using this method. The nucleic acid sequence can be anylength. For example, the nucleic acid sequence can be at least 10, atleast 50, at least 100, at least 150, at least 200, at least 250, atleast 300, at least 400 or at least 500 nucleotides in length. Thenucleic acid sequence can be naturally occurring or artificial. Forinstance, the method may be used to verify the sequence of amanufactured oligonucleotide. The methods are typically carried out invitro.

The methods may be carried out using any suitable membrane/pore systemin which a pore comprising a construct of the invention is inserted intoa membrane. The methods are typically carried out using (i) anartificial membrane comprising a pore comprising a construct of theinvention, (ii) an isolated, naturally occurring membrane comprising apore comprising a construct of the invention, or (iii) a cell expressinga pore comprising a construct of the invention. The methods arepreferably carried out using an artificial membrane. The membrane maycomprise other transmembrane and/or intramembrane proteins as well asother molecules in addition to the pore of the invention.

The membrane forms a barrier to the flow of ions, nucleotides andnucleic acids. The membrane is preferably a lipid bilayer. Lipidbilayers suitable for use in accordance with the invention can be madeusing methods known in the art. For example, lipid bilayer membranes canbe formed using the method of Montal and Mueller (1972). Lipid bilayerscan also be formed using the method described in InternationalApplication No. PCT/GB08/000563.

The methods of the invention may be carried out using lipid bilayersformed from any membrane lipid including, but not limited to,phospholipids, glycolipids, cholesterol and mixtures thereof. Any of thelipids described in International Application No. PCT/GB08/000563 may beused.

Methods are known in the art for inserting pores into membranes, such aslipid bilayers. Some of those methods are discussed above.

Interaction Between the Pore and Nucleotides

The nucleotide or nucleic acid may be contacted with the pore on eitherside of the membrane. The nucleotide or nucleic acid may be introducedto the pore on either side of the membrane. The nucleotide or nucleicacid is typically contacted with the side of the membrane on which theenzyme is attached to the pore. This allows the enzyme to handle thenucleic acid during the method.

A proportion of the nucleotides of the target nucleic acid sequenceinteracts with the pore and/or adaptor as it passes across the membranethrough the barrel or channel of the pore. Alternatively, if the targetsequence is digested by an exonuclease, the nucleotide may interact withthe pore via or in conjunction with the adaptor, dissociate from thepore and remain on the same side of the membrane. The methods mayinvolve the use of pores in which the orientation of the adaptor isfixed. In such embodiments, the nucleotide is preferably contacted withthe end of the pore towards which the adaptor is oriented. Mostpreferably, the nucleotide is contacted with the end of the pore towardswhich the portion of the adaptor that interacts with the nucleotide isorientated.

The nucleotides may interact with the pore in any manner and at anysite. As discussed above, the nucleotides preferably reversibly bind tothe pore via or in conjunction with the adaptor. The nucleotides mostpreferably reversibly bind to the pore via or in conjunction with theadaptor as they pass through the pore across the membrane. Thenucleotides can also reversibly bind to the barrel or channel of thepore via or in conjunction with the adaptor as they pass through thepore across the membrane.

During the interaction between a nucleotides and the pore, thenucleotide affects the current flowing through the pore in a mannerspecific for that nucleotide. For example, a particular nucleotide willreduce the current flowing through the pore for a particular mean timeperiod and to a particular extent. In other words, the current flowingthrough the pore is distinctive for a particular nucleotide. Controlexperiments may be carried out to determine the effect a particularnucleotide has on the current flowing through the pore. Results fromcarrying out the method of the invention on a test sample can then becompared with those derived from such a control experiment in order toidentify a particular nucleotide.

Apparatus

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore comprising aconstruct of the invention is inserted into a membrane. The methods maybe carried out using any apparatus that is suitable for stochasticsensing. For example, the apparatus comprises a chamber comprising anaqueous solution and a barrier that separates the chamber into twosections. The barrier has an aperture in which the membrane containingthe pore is formed. The nucleotide or nucleic acid may be contacted withthe pore by introducing the nucleic acid into the chamber. The nucleicacid may be introduced into either of the two sections of the chamber,but is preferably introduced into the section of the chamber containingthe enzyme.

The methods may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562.

The methods involve measuring the current passing through the poreduring interaction with the nucleotides. Therefore the apparatus alsocomprises an electrical circuit capable of applying a potential andmeasuring an electrical signal across the membrane and pore. The methodsmay be carried out using a patch clamp or a voltage clamp. The methodspreferably involves the use of a voltage clamp.

Conditions

The methods of the invention involve the measuring of a current passingthrough the pore during interaction with nucleotides in a target nucleicacid sequence. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExamples. The method is carried out with a voltage applied across themembrane and pore. The voltage used is typically from −400 mV to +400mV. The voltage used is preferably in a range having a lower limitselected from −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20mV and 0 mV and an upper limit independently selected from +10 mV, +20mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltageused is more preferably in the range 120 mV to 170 mV. It is possible toincrease discrimination between different nucleotides by a pore of theinvention by using an increased applied potential.

The methods are carried out in the presence of any alkali metal chloridesalt. In the exemplary apparatus discussed above, the salt is present inthe aqueous solution in the chamber. Potassium chloride (KCl), sodiumchloride (NaCl) or caesium chloride (CsCl) is typically used. KCl ispreferred. The salt concentration is typically from 0.1 to 2.5M, from0.3 to 1.9M, from 0.5 to 1.8M, from 0.7 to 1.7M, from 0.9 to 1.6M orfrom 1M to 1.4M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.However, lower salt concentrations are preferably used so that theenzyme is capable of functioning. The salt concentration is preferablyfrom 150 to 500 mM. Good nucleotide discrimination at these low saltconcentrations can be achieved by carrying out the method attemperatures above room temperature, such as from 30° C. to 40° C.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the methods.One suitable buffer is Tris-HCl buffer. The methods are typicallycarried out at a pH of from 4.0 to 10.0, from 4.5 to 9.5, from 5.0 to9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5.The pH used is preferably about 7.5.

The methods are typically carried out at from 0° C. to 100° C., from 15°C. to 95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C.to 80° C., 19° C. to 70° C., or from 20° C. to 60° C. The methods may becarried out at room temperature. The methods are preferably carried outat a temperature that supports enzyme function, such as about 37° C.Good nucleotide discrimination can be achieved at low saltconcentrations if the temperature is increased.

In addition to increasing the solution temperature, there are a numberof other strategies that can be employed to increase the conductance ofthe solution, while maintaining conditions that are suitable for enzymeactivity. One such strategy is to use the lipid bilayer to divide twodifferent concentrations of salt solution, a low salt concentration ofsalt on the enzyme side and a higher concentration on the opposite side.One example of this approach is to use 200 mM of KCl on the cis side ofthe membrane and 500 mM KCl in the trans chamber. At these conditions,the conductance through the pore is expected to be roughly equivalent to400 mM KCl under normal conditions, and the enzyme only experiences 200mM if placed on the cis side. Another possible benefit of usingasymmetric salt conditions is the osmotic gradient induced across thepore. This net flow of water could be used to pull nucleotides into thepore for detection. A similar effect can be achieved using a neutralosmolyte, such as sucrose, glycerol or PEG. Another possibility is touse a solution with relatively low levels of KCl and rely on anadditional charge carrying species that is less disruptive to enzymeactivity.

Exonuclease-Based Methods

In one embodiment, the method of sequencing a target nucleic acidsequence involves contacting the target sequence with a pore having anexonuclease enzyme, such as deoxyribonuclease, attached thereto. Theconstructs needed to make such pores are discussed above. Any of theexonuclease enzymes discussed above may be used in the method. Theexonuclease releases individual nucleotides from one end of the targetsequence. Exonucleases are enzymes that typically latch onto one end ofa nucleic acid sequence and digest the sequence one nucleotide at a timefrom that end. The exonuclease can digest the nucleic acid in the 5′ to3′ direction or 3′ to 5′ direction. The end of the nucleic acid to whichthe exonuclease binds is typically determined through the choice ofenzyme used and/or using methods known in the art. Hydroxyl groups orcap structures at either end of the nucleic acid sequence may typicallybe used to prevent or facilitate the binding of the exonuclease to aparticular end of the nucleic acid sequence.

The method involves contacting the nucleic acid sequence with theexonuclease so that the nucleotides are digested from the end of thenucleic acid at a rate that allows identification of a proportion ofnucleotides as discussed above. Methods for doing this are well known inthe art. For example, Edman degradation is used to successively digestsingle amino acids from the end of polypeptide such that they may beidentified using High Performance Liquid Chromatography (HPLC). Ahomologous method may be used in the present invention.

The rate at which the exonuclease functions is typically slower than theoptimal rate of a wild-type exonuclease. A suitable rate of activity ofthe exonuclease in the method of sequencing involves digestion of from0.5 to 1000 nucleotides per second, from 0.6 to 500 nucleotides persecond, 0.7 to 200 nucleotides per second, from 0.8 to 100 nucleotidesper second, from 0.9 to 50 nucleotides per second or 1 to 20 or 10nucleotides per second. The rate is preferably 1., 10, 100, 500 or 1000nucleotides per second. A suitable rate of exonuclease activity can beachieved in various ways. For example, variant exonucleases with areduced optimal rate of activity may be used in accordance with theinvention.

Pushing or Pulling DNA Through the Pore

Strand sequencing involves the controlled and stepwise translocation ofnucleic acid polymers through a pore. The majority of DNA handlingenzymes are suitable for use in this application provided theyhydrolyse, polymerise or process single stranded DNA or RNA. Preferredenzymes are polymerases, exonucleases, helicases and topoisomerases,such as gyrases. The enzyme moiety is not required to be in as close aproximity to the pore lumen as for individual nucleotide sequencing asthere is no potential for disorder in the series in which nucleotidesreach the sensing moiety of the pore.

The two strategies for single strand DNA sequencing are thetranslocation of the DNA through the nanopore, both cis to trans andtrans to cis, either with or against an applied potential. The mostadvantageous mechanism for strand sequencing is the controlledtranslocation of single strand DNA through the nanopore with an appliedpotential. Exonucleases that act progressively or processively on doublestranded DNA can be used on the cis side of the pore to feed theremaining single strand through under an applied potential or the transside under a reverse potential. Likewise, a helicase that unwinds thedouble stranded DNA can also be used in a similar manner. There are alsopossibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

Kits

The present invention also provides kits for producing a modified porefor use in sequencing nucleic acids. In one embodiment, the kitscomprise at least one construct of the invention and any remainingsubunits need to form a pore. The kits may comprise enough constructs ofthe invention to form a complete pore (i.e. a homo-oligomer). The kitsmay comprise any of the constructs and subunits discussed above. Apreferred kit comprises (i) a construct comprising a subunit comprisingthe sequence shown in SEQ ID NO: 2 or a variant thereof and (ii) sixsubunits comprising the sequence shown in SEQ ID NO: 2 or a variantthereof. A more preferred kit comprises (i) a construct comprising thesequence shown in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 30 or a variantthereof and (ii) six subunits comprising the sequence shown in SEQ IDNO: 2 or a variant thereof.

In another embodiment, the kits comprise at least one polynucleotidesequence of the invention and polynucleotide sequences encoding anyremaining subunits needed to form a pore. The kit may comprise enoughpolynucleotides of the invention to encode a complete pore (i.e. ahomo-oligomer). The kits may comprise any of the polynucleotidesdescribed above. A preferred kit comprises (i) a polynucleotide sequenceencoding a construct, which comprises a subunit comprising the sequenceshown in SEQ ID NO: 2 or a variant thereof and (ii) six polynucleotidesequences each encoding a subunit comprising the sequence shown in SEQID NO: 2 or a variant thereof. A more preferred kit comprises (i) apolynucleotide sequence encoding a construct comprising the sequenceshown in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 30 or a variant thereofand (ii) six polynucleotide sequences each encoding a subunit comprisingthe sequence shown in SEQ ID NO: 2 or a variant thereof.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotidesequences, a membrane as defined above or voltage or patch clampapparatus. Reagents may be present in the kit in a dry state such that afluid sample resuspends the reagents. The kit may also, optionally,comprise instructions to enable the kit to be used in the method of theinvention or details regarding which patients the method may be usedfor. The kit may, optionally, comprise nucleotides.

The following Example illustrates the invention:

Example

1 Materials and Methods

1.1 Bacterial Strains and Growth Conditions

The bacterial strains used in this work were E. coli strains XL-10 Goldand BL21 DE3 pLysS (Stratagene). E. coli strains were grown at 370Ceither in Luria-Bertani Broth (LB), Terrific Broth at 225 rpm,Luria-Bertani agar (LA) or tryptone-yeast extract agar (TY) (Bertani. G.(1951). Studies on lysogenesis. I. The mode of phage liberation bylysogenic Escherichia coli. Journal of Bacteriology. 62, 293-300;Beringer, J. (1974). R factor transfer in Rhizobium leguminosarum.Journal of General Microbiology. 84, 188-98; and Tartoff, K. and Hobbs,C. (1987). Improved media for growing plasmid and cosmid clones.Bethesda Research Labs Focus. 9, 12). Antibiotics were used at thefollowing concentrations: Ampicillin 100 μg ml⁻¹; chloramphenicol 30 μgml⁻¹.

1.2 Genetic Manipulations

All general DNA cloning was performed as adapted methods of thatpreviously described (Sambrook, J. and Russell, D. (2001). MolecularCloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). DNA polymerases, restrictionendonucleases, exonuclease, ligases and phosphatases were all obtainedfrom New England Biolabs. Exonuclease genes were manufactured byGenScript Corporation and received as fragments cloned into pT7-SC1, byBspEI or NdeI/HindIII. All mutations and fusion constructs wereassembled in the expression vector pT7-SC1 (Cheley, S., Malghani, M.,Song, L., Hobaugh, M., Gouaux, E., Yang, J. and Bayley, H. (1997).Spontaneous oligomerization of a staphylococcal alpha-hemolysinconformationally constrained by removal of residues that form thetransmembrane beta-barrel. Protein Engineering. 10, 1433-43) andverified by sequencing using either the T7 forward or reverse primers,EcoExoIII_seq and EcoExoI_seq.

Site directed mutagenesis of the αHL gene was performed by in vivohomologous recombination of PCR products (Jones, D. (1995) PCRmutagenesis and recombination in vivo. In PCR primer: a laboratorymanual. In: Dveksler, C. (ed). Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). Amplification of two halves of the target plasmidwith complimentary primer pairs generates two PCR products withcomplimentary sequences at both the 5′ and 3′ ends. Transformation ofboth products into chemically competent E. coli allows in vivohomologous recombination. For all mutagenesis SC46 was used as theantisense primer for amplification of product 1 and SC47 as the senseprimer for amplification of product 2. These complementary primerbinding sites are within the β-lactamase gene of pT7-SC1. Coloniesrecovered on LA 100 ng μl⁻¹ ampicillin therefore indicated successfulhomologous recombination.

PCR was conducted in 50 μl reactions using 1 unit Phusion™ DNApolymerase, 0.2 mM dNTPs, 1 μM primers and 4 ng BamHI/HindIII orNdeI/EcoNI digested plasmid DNA. Reactions were cycled as follows: 1cycle of 98° C. for 2 min; 30 cycles of 98° C. for 15 s, 57° C. for 30 sand 72° C. for 45 s; and a final extension of 72° C. for 5 min. 2.5 μlof each pair of PCR products were mixed and used to transform chemicallycompetent E. coli (XL-10 Gold).

1.3 Rapid In Vitro Transcription Translation

[³⁵S]L-methionine labelled proteins were generated by coupled in vitrotranscription and translation (IVTT) using an E. coli T7-S30 extractsystem for circular DNA (Promega). The complete amino acid mixture (1mM) minus cysteine and the complete amino acid mixture (1 mM) minusmethionine, supplied in the kit, were mixed in equal volumes to obtainthe working amino acid solution required to generate high concentrationsof the protein. Reactions were scaled up or down based on the following,for a 50 μl reaction volume: 20 μl S30 Premix solution; 5 μl amino acidmix; 1 μl [³⁵S]L-methionine (MP Biomedicals. 1175 Ci mmol⁻¹, 10 mCiml⁻¹), 1 μl rifampicin (0.8 mg ml⁻¹), 8 μl plasmid DNA (400 ng μl⁻¹) and15 μl. T7 S30 extract. Synthesis was carried out for 1.5 hours at 37° C.to produce 50 μl of radiolabelled IVTT protein. Different proteins werealso co-expressed in one reaction as for coupled transcription,translation and oligomerisation. The reaction components remained thesame except the DNA concentration was divided accordingly for eachplasmid encoding each protein. Protein samples were centrifuged at14,000 rpm for 10 minutes to separate insoluble debris of IVTTreactions.

1.4 In Vivo Protein Expression

Wild-type α-hemolysin and fusion constructs were cloned into theexpression vector pT7-SC1, under the control of the inducible T7promoter, and expressed in E. coli (BL21 DE3 pLysS. Stratagene) assoluble proteins. Cultures were grown to a high OD₆₀₀ (approximately1.5-2) at 37° C. and 240 rpm in Terrific broth medium (100 μg μl⁻¹ampicillin and 30 μg l⁻¹ chloramphenicol). The temperature was reducedto 18° C. and cultures left for 30 minutes to equilibrate. Overexpression of the target protein was induced by addition of IPTG to themedium (0.2 mM). After 18 hours cells were pelleted at 10,000 rpm for 30minutes at 4° C. Cells were resuspended and lysed by the addition ofBugBuster (Novagen) supplemented with the addition of benzonase,EDTA-free proteinase inhibitors (Roche) and to 50 mM MgCl₂. Cell debriswas pelleted by centrifugation at 10.000 rpm for 30 minutes at 4° C. andpolyethyleneimine (PEI) added to the supernatant. The recoveredsupernatant was incubated for 30 mins at 4° C. after which precipitatewas removed by centrifugation at 10,000 rpm for 30 minutes at 4° C.Clarified lysate was filtered and adjusted to pH 8.0, 500 mM NaCl, 10 mMImidazole.

His-tagged proteins were purified as standard practice by Ni-NTAaffinity chromatography and gel filtration. Non-tagged α-hemolysinsubunits were purified as standard practice by cation exchange followedby gel filtration.

1.4.1 Affinity Purification (His-Tag)

Clarified lysate was filtered and adjusted to pH 8.0, 500 mM NaCl, 10 mMImidazole before loading onto a His-Trap crude column (GE Healthcare)and eluted with 300 mM Imidazole. Fractions containing the protein ofinterest were combined and applied to a gel filtration columnequilibrated with 10 mM TRIS pH 8.0, 100 mM NaCl, 1 mM DTT. Elutedprotein was evaluated by SDS-PAGE.

1.4.2 Ion Exchange

Clarified lysate was filtered and adjusted to 10 mM MES pH 6.0 beforeloading onto a cation exchange column (GE Healthcare) and eluting with0-500 mM NaCl. Fractions containing the protein of interest werecombined and applied to a gel filtration column. Eluted protein wasevaluated by SDS-PAGE.

To maintain the reactivity of engineered cysteine residues inα-Hemolysin derivatives, required as sites for chemical modification,proteins were purified using the same buffers but supplemented to 1 mMDTT. Exonucleases or exonuclease fusion proteins were purified using thesame buffers supplemented to 1 mM MgCl₂.

1.5 Oligomerisation on Red Blood Cell Membranes

α-Hemolysin monomers were mixed in various molar ratios and allowed tooligomerise on rabbit erythrocyte membranes (2.5 mg protein ml⁻¹) for 1hour at either room temperature, 30° C., 37° C. (or 42° C. After theincubation, reaction mixture was centrifuged at 14.000 rpm for 10minutes and supernatant discarded. Membrane pellet was washed byresuspension in 200 μl MBSA (10 mM MOPS, 150 mM NaCl, pH 7.4 containing1 mg ml⁻¹ bovine serum albumin) and centrifuging again at 14,000 rpm for10 minutes. After discarding the supernatant, membrane pellet wasdissolved in 75 μl of IX Laemmli sample buffer, with the addition ofβ-mercaptoethanol. The entire sample was loaded into a single well of a5% SDS-polyacrylamide gel and elelctrophoresed for ˜18 hours at 50 V,with 0.01 mM sodium thioglycolate included in the running buffer. Gelwas vacuum-dried onto a Whatman 3 mm filter paper at 50° C. for aboutthree hours and exposed to an X-ray film overnight (Kodak). The oligomerband was excised from the gel, using the autoradiogram as template, andthe gel slice rehydrated in 300 μl TE buffer (10 mM Tris, 1 mM EDTA, pH8.0) containing 2 mM DT. After removing the Whatman filter paper slice,gel piece was crushed using a sterile pestle. Oligomer protein wasseparated from gel debris by centrifuging through 0.2 UM celluloseacetate microfilterage tubes (Rainin) at 14.000 rpm for 30 min. Filtratewas stored in aliquots at −80° C.

1.6 Oligomerisation on Synthetic Lipid Vesicles

Synthetic lipid vesicles composed of: 30% cholesterol; 30%phosphatidylcholine (PC); 20% phosphatidylethanolamine (PE); 10%sphingomyelin (SM); 10% phosphatidylserine (PS); were prepared by bathsonication for 15 minutes at room temperature. Organic solvent isevaporated by a gentle stream of nitrogen until a dry film is produced.Deionised water added to give a required concentration of 2.5 mg ml⁻¹and mixture bath sonicated again for 15 minutes. Wild-type α-hemolysinand fusion monomers were mixed in various molar ratios and allowed tooligomerise on synthetic lipid vesicles (2.5 mg ml⁻¹ for every 1 mgα-hemolysin monomer) for 1 hour at either room temperature, 30° C., 37°C. or 42° C. and 350 rpm. To pellet lipid associated proteins sampleswere centrifuged at 14,000 rpm for 10 minutes. Pellet was washed once inMBSA (10 mM MOPS, 150 mM NaCl, pH 7.4 containing 1 mg ml⁻¹ bovine serumalbumin) and lipids were dissolved by addition of 0.1-1%n-Dodecyl-D-maltopyranoside (DDM), for 1 hour at either 4° C. or roomtemperature. To purify the fusion homo and heteroheptamers away fromwild-type homoheptamer 300 μl of Ni-NTA agarose (Qiagen) was added andleft overnight at 4° C. and 350 rpm. Affinity bound heptamer was peltedwith Ni-NTA agarose by centrifugation at 14,000 rpm for 10 minutes. TheNi-NTA agarose beads were washed twice in 500 μl wash buffer (10 mMTris, 10 mM Imidazole, 500 mM NaCl, pH 8.0) for 10 minutes and recoveredby centrifugation. Purified heteroheptamer was eluted in 500 μl elutionbuffer (10 mM Tris, 250 mM Imidazole. pH 8.0) for 1 hour at 4° C. TheNi-NTA agarose was removed by centrifugation and the supernatantcontaining the eluted purified fusion heptamers removed. Elutedheptamers were de-salted by passage through a buffer exchange column(NAP-5, GE Healthcare), equilibrated with 10 mM Tris pH 8.0.

1.7 Exonuclease Fluorescence Assay

Recombinant E. coli Exonuclease III was purchased from New EnglandBiolabs (100 units μl⁻¹). Double stranded DNA template labelled with a5′ fluorophore (5HEX) on the sense strand and a 3′ black hole quencher(BHQ-2a-Q) on the antisense strand was obtained from Operon.

The oligo sequences are given below along with the respectivefluorophore and quencher pair:

(SEQ ID NO: 31) 5′[5HEX]GCAACAGAGCTGATGGATCAAATGCATTAGGTAAACATGTTACGTCGTAA 3′ (SEQ ID NO: 32)5′CGATCTTACGACGTAACATGTTTACCTAATGCATTTGATCCATCAGCT CTGTTGC[BHQ2a]3′The substrate dsDNA has a 5 bp overhang at the 5′ end of the antisensestrand, enabling initiation of exonuclease III on the 3′ end of thesense strand.

Fluorescence measurements were taken using a Cary Eclipse (Varian) withan excitation and emission wavelength of 535 and 554 nm respectively andan excitation and emission slit of 5 nm. Measurements were taken every 4seconds for 60 minutes. 40 μl reactions were performed at 37° C. andconsisted of: 200 nm substrate dsDNA; 25 mM Tris pH 7.5; 1 mM MgCl₂; 100mM KCl; 0.001 units Exo III; unless otherwise stated.

1.8 Planar Bilayer Recordings

All bilayers were formed by apposition of two monolayers of1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) acrossa 60-150 μm diameter aperture in Teflon film (25 μm thickness fromGoodfellow, Malvern, Pa.), which divided a chamber into two buffercompartments (cis and trans) each with a volume of 1 ml. Bilayers wereformed across the aperture by consecutively raising the buffer level ineach compartment until a high resistance seal was observed (≥10 GΩ).Unless otherwise stated, fusion heptamers and DNA or dNMPs were added tothe cis compartment, which was connected to ground. The adapter moleculeam7βCD or am6-amPDP1-βCD was added to the trans compartment if required,which was connected to the head-stage of the amplifier. Unless statedotherwise, experiments were carried out in 25 mM Tris.HCl, 400 mM KCl pH8.0, at 22° C.

1.9 Exonucleases

Exonucleases, such as deoxyribonucleases, are a subgroup of the EC 3.1enzymes. They catalyse the hydrolysis of the phosphodiester bond betweenadjacent bases in a DNA strand to release individual nucleoside 5′mono-phosphates (FIG. 1). Attractive activities catalyse the cleavage ofthis bond (through nucleophilic attack of an activated water moleculeupon the phosphorus) as shown.

There are a limited number of distinct enzymatic activities that degradenucleic acids into their component parts, although numerous homologueswill exist in different organisms (for example, Exonuclease III). From adetailed literature search, the two most processive exonuclease enzymesare Exonuclease I, encoded by the sbcB gene of E. coli, andλ-exonuclease, encoded by the exo gene of bacteriophage λ (Thomas, K.and Olivera, B. (1978) Processivity of DNA exonucleases. Journal ofBiological Chemistry. 253, 424-429; and Zagursky, R. and Hays, J.(1983). Expression of the phage lambda recombination genes exo and betunder lacPO control on a multi-copy plasmid. Gene. 23, 277-292). Inaddition, activity of Exonuclease I has been demonstrated in high saltconcentrations (Hornblower, B., Coombs, A., Whitaker, R., Kolomeisky,A., Picone, S., Meller, A. Akeson, M. (2007). Single-molecule analysisof DNA-protein complexes using nanopores. Nature Methods. 4, 315-317).As λ exonuclease is a trimer the attachment of a functional exonucleaseis more challenging so the monomeric enzyme Exonuclease III was alsoincluded, as despite its shorter processivity rate it also degrades onestrand of dsDNA to yield nucleoside 5′ monophosphates. Whilst Exo Idegrades ssDNA in a 3′-5′ direction RecJ acts 5′-3′ and so was alsoincluded in this work (Lovett, S. and Kolodner, R. (1989).Identification and purification of a single-stranded-DNA-specificexonuclease encoded by the recJ gene of Escherichia coli. Proceedings ofthe National Academy of Sciences of the United States of America. 86,2627-2631). Both ssDNA exonucleases have been demonstrated to interactand act cooperatively with single stranded binding protein (Genschel,J., Curth, U. and Urbanke, C. (2000) Interaction of E. colisingle-stranded DNA binding protein (SSB) with exonuclease I. Thecarboxy terminus of SSB is the recognition site for the nuclease.Biological Chemistry. 381, 183-192; and Han, E., Cooper, D., Persky, N.,Sutera, V., Whitaker, R., Montello, M. and Lovett, S. (2006). RecJexonuclease: substrates, products and interaction with SSB. NucleicAcids Research. 34, 1084-1091). The use of these proteins may berequired to prevent secondary structure formation of the ssDNA substratethat may enzyme initiation or processivity in high salt concentrations.

Four exonucleases are used in this Example:

1. Exo III from E. coli, Monomeric, dsDNA, 3′-5′ (SEQ ID NOs: 9 and 10)

2. Exo I from E. coli. Monomeric, ssDNA, 3′-5′ (SEQ ID NOs: 11 and 12)

3. RecJ from T. thermophilus, Monomeric, ssDNA, 5′-3′ (SEQ ID NOs: 13and 14)

4. λ Exo from λ bacteriophage, Trimeric, dsDNA, 5′-3′ (the sequence ofone monomer is shown in SEQ ID NOs: 15 and 16)

High resolution crystal structures are available for all these enzymes(Mol, C., Kuo, C., Thayer, M., Cunningham, R. and Tainer, J. (1995)Structure and function of the multifunctional DNA-repair enzymeexonuclease III. Nature. 374, 381-386; Kovall, R. and Matthews, B.(1997). Toroidal structure of lambda-exonuclease. Science. 277,1824-1827; and Busam, R. (2008). Structure of Escherichia coliexonuclease I in complex with thymidine 5′-monophosphate. ActaCrystallographica. 64, 206-210) and are shown in FIG. 2. The TthRecJ isthe enzymes core domain as identified by Yamagata et al. (Yamagata, A.,Masui, R., Kakuta, Y., Kuramitsu, S. and Fukuyama, K. (2001).

1.10 Genetic Attachment

Taking the characteristics of the exonuclease as detailed above, thework described here was guided by the generation of a hypothetical modelin which just one of the seven subunits of the αHL heptamer is modifiedto carry the exonuclease activity. FIG. 3 is a representation of thefusion construct assembled into a heteroheptamer with the exonucleaseattached to a loop on the cis side of the protein. This model satisfiesother additional desirable characteristics. An exonuclease fused on thecis side of the αHL heptamer under positive potential should releasemonophosphate nucleosides or ssDNA that will migrate from the cis to thetrans side of the pore. This direction of migration is standard in muchof the published literature of nanopore sensing. The genetic attachmentof an exonuclease within a loop region also invariably means that the Nand C terminal linkers can be designed to limit and constrain themobility of the exonuclease in relation to the lumen of the pore.

In order to create a genetic fusion of the α-HL and the exonucleaseproteins, genetic manipulation of the pre-existing expression plasmidpT7-SC1 carrying the wild-type α-HL gene was made (SEQ ID NO: 3). Thisplasmid carries the gene encoding the wild-type α-HL (SEQ ID NO: 1)without the benefit of any mutations that have been demonstrated toenhance the capacity of the pore to detect and discriminatemonophosphate nucleosides. Unique BspEI restriction endonuclease siteswere engineered into the α-HL gene at three specific locations, toenable insertion of the exonuclease gene, detailed below. Three plasmidsare thus generated, with each one carrying just a single BspEI site forexonuclease gene infusion.

The first insertion site, L1, is located between residues T18 and T19 ofthe first loop region (N6-V20) of the α-hemolysin protein (SEQ ID NO:6). The second insertion site, L2, is located between residues D44 andD45 of the start of the second loop region (D44-K50) of the α-hemolysinprotein (SEQ ID NO: 7). The third insertion site, L2b, is locatedbetween residues K50 and K51 of the end of the second loop region(D44-K50) of the α-hemolysin protein (SEQ ID NO: 8).

Exonuclease genes were codon optimised for expression in E. coli andsynthesised by GenScript Corporation (SEQ ID NOs: 10, 12, 15 and 16).Genes were flanked by regions encoding 10 residues of repeatingserine-glycine. Such a protein sequence is believed to be substantiallydevoid of a defined secondary or tertiary structure. The terminal endsof the linkers were also defined by recognition sequences for therestriction endonuclease BspEI, as this sequence also encodes a serineand glycine that form part of the linker. The recognition site of thisenzyme (TCCGGA) was similarly engineered into the three specificlocations within the αHL gene to provide a means of inserting theexonuclease genes in frame at these defined locations.

The recombinant gene encodes a fusion protein consisting of: a portionof αHL; a 10 serine-glycine linker region; an exonuclease; a 10serine-glycine linker region; and the remaining portion of αHL. Oncemade, the chimeric gene construct was sequenced and verified to be asshown in FIG. 4.

Both the N and C-termini of α-hemolysin are suitable for genetic fusionto an enzyme. It has been shown that the 17 N-terminal residues, whichconstitute the amino latch, are dispensable for heptamer formation.Whilst it is not possible to delete more than 3 residues from theC-terminus, without effecting oligomerisation, it is already readilypresented as a possible attachment point at the back of the cap domain(Walker, B. and Bayley. H. (1995). Key residues for membrane binding,oligomerization and pore-forming activity of Staphylococcal α-hemolysinidentified by cysteine scanning mutagenesis and targeted chemicalmodification. The Journal of Biological Chemistry. 270, 23065-23071).

The attachment of enzymes at the N and C-terminus of α-hemolysin wascarried out in a similar manner to that described above. The enzyme andα-hemolysin domains were again mediated by serine-glycine rich linkersto ensure the physical separation necessary for correct folding andspatial separation of each protein domain. The exact details ofattachment are however detailed in a later section.

The hemolysin monomers were initially used as a wildtype monomer (wt),however we have shown that a HL-M113R/N139Q monomer shows improved basediscrimination and the baseline was changed to this background. Furtherwork showed that the base best resolution was achieved when an adaptermolecule was attached to the L135C position, this was added to thehemolysin-exonuclease fusion in later constructs.

In the construct nomenclature, the monomer HL-M113R/N139Q is abbreviatedto HL-RQ and the HL-M113R/N139Q/L135C monomer is abbreviated to HL-RQC.Therefore the fusion constructHL-(M113R/N139Q)₆(M113R/N139Q/L135C-EcoExoIII-L1-H6)₁ is shortened toHL-(RQ)₆(RQC-EcoExoIII-L1-H6)₁.

2 Results

2.1 Oligomerisation of Loop 1 Fusion Proteins

Water soluble α-hemolysin monomers can bind to and self-assemble on alipid membrane to form a transmembrane pore of defined structure, via anintermediate heptameric prepore (Walker, B. and Bayley, H. (1995). Keyresidues for membrane binding, oligomerization and pore-forming activityof Staphylococcal α-hemolysin identified by cysteine scanningmutagenesis and targeted chemical modification. The Journal ofBiological Chemistry. 270, 23065-23071). Fully assembled pores can thenbe isolated and recovered through SDS PAGE, for biophysicalcharacterisation. Radiolabelled α-hemolysin monomers produced through invitro transcription translation (IVTT) and oligomerised on purifiedrabbit red blood cell membranes, enable heptamers to be recovered fromthe gel using the autoradiograph as template. Modified monomers can alsobe incorporated into the heptamer in any number and at any of thesubunit positions (1-7). The modified subunit also typically carries apoly-aspartate tail to allow the differential migration of homo orheteroheptamers on SDS PAGE for ease of purification for each variant(Braha, O., Walker, B., Cheley, S., Kasianowicz, J., Song, L., Gouaux,J. and Bayley, H. (1997). Designed protein pores as components forbiosensors. Chemistry and Biology. 4, 497-505). Due to the size of theexonuclease proteins it was not expected that a poly-aspartate tailwould be required on the fusion monomers, as the exonuclease aloneshould cause a significant shift in electrophoretic mobility to enableidentification of individual heteroheptamers away from wild-typehomoheptamer.

To determine if a mixture of HL-RQ and fusion monomers were able to formheteroheptamers [³⁵S]L-methionine labelled HL-RQ and fusion proteins(HL-wt-EcoExoIII-L1-H6 (SEQ ID NO: 18). HL-RQC-EcoExoIII-L1-H6 (SEQ IDNO: 20), HL-RQC-EcoExoI-L1-H6 (SEQ ID NO: 22) and HL-RQC-TthRecJ-L1-H6(SEQ ID NO: 24) were expressed by IVTT and oligomerised on purifiedrabbit red blood cell membranes. The autoradiograph of the gelidentified several putative heptamer bands of differing size for allenzyme fusions (FIG. 5).

To characterise these heptamer bands and to identify the ratio ofsubunits within each, proteins were excised from the gel. Heatingheptamer at 95° C. for 10 minutes breaks the protein into itsconstitutive monomers, which can then be visualised on SDS PAGE fordensitometry to determine the heptamer subunit composition. Thedifferent characteristic heptamer bands can then be identified as homoor heteroheptamers that consist of different ratios of wild-type andfusion α-HL monomers. This characterisation was performed for putativeheptamer bands generated using both the HL-wt-EcoExoIII-L1-H6 andHL-RQC-EcoExoI-L1-H6 fusion proteins.

An importance for a sequencing application is that there preferentiallybe only one exonuclease moiety, ensuring bases are released only from asingle DNA stand being processed at any one time. Electrophoreticmigration of a 6:1 HL-monomer:HL-Exonuclease species away from otheroligomers is therefore desired for ease of purification. Surprisingly,the HL-(RQ)₆(wt-EcoExoI-L1-H6)₁ heptamer migrates to a position slightlylower down the gel than HL-(RQ)₇, despite the presence of a ˜36 kDaexonuclease being present on one of the subunits. This band also has a“doublet” appearance, possibly caused by incorrect incorporation of thefusion subunits amino latch due to the downstream insertion of theexonuclease in loop 1 or translation initiating at two points (the startof the fusion protein at hemolysin M1 and also at the first methionineof ExoIII) giving a mixed pool of fusion proteins. The EcoExoIII fusionprotein gives formation of all theoretical heteroheptamer varieties andthe wild-type and fusion protein homoheptamers. As a significantlysmaller protein, ˜36 kDa, and with its N and C terminus co-localised itis perhaps unsurprising that EcoExoIII performs better than EcoExoI orTthRecJ as an exonuclease suitable for inserting into loop regions togive good heteroheptamer formation. Both the EcoExoI and TthRecJ fusionproteins give still show formation of heteroheptamers, although with alimited number of fusion monomer subunits, but in contrast the 6:1heteroheptamer of EcoExoIII these 6:1 heteroheptamers migrate to aposition identical to HL-(RQ)₇.

It is an important consideration that by varying the ratio of wild-typeto fusion monomer different bands corresponding to the different homoand heteroheptamers were observed. This allows the control of homo orheteroheptamer formation based on the molar ratio of different monomersubunits, which is important for the preferential generation of HL-(RQ)₆(RQ-Exonuclease-H6)₁ (FIG. 6).

The conditions for the HL-(RQ)₆(wt-EcoExoIII-L1-H6)₁ heteroheptamerformation were optimised by varying the ratios of monomer proteins. Apreferred ratio of 100:1 gives predominately formation of one type ofheteroheptamer, HL-(RQ)₆(wt-EcoExoIII-L1-H6)₁, as well as wild-typehomoheptamer, HL-(RQ)₇. Affinity purification by the hexa-His tag of thefusion subunit then allows separation of heteroheptamer from HL-RQhomoheptamer.

The HL-(wt-EcoExoIII-L1-H6)₇ homoheptamer and theHL-(RQ)₆(wt-EcoExoIII-L1-H6)₁ heteroheptamer bands were excised from thegel and the protein pores recovered by re-hydration and maceration ofthe gel slice. These isolated heptamers were both able to insert intoplanar lipid bilayers to give single channel recordings. The singlechannel trace for the HL-(wt-EcoExoIII-L1-H6)₇ homoheptamer, however,exhibited numerous blocking events at ≥80 mV. This could be attributedto the presence of seven denatured exonuclease peptide chainssurrounding the cap domain, as these events were significantly lesspronounced with the HL-(RQ)₆(wt-EcoExoIII-L1-H6)₁ heteroheptamer. TheHL-(RQ)₆(wt-EcoExoIII-L1-H6) heteroheptamer gave an open pore current of˜160 pA and a heteroheptamer containing the mutations necessary for basediscrimination HL-(RQ)₆(RQC-EcoExoIII-L1-H6)₁ showed covalent attachmentof the β-cyclodextrin adapter molecule, which is characterised by anpersistent current block to ˜90 pA.

The construction of a fusion protein involves the linking of twoproteins or domains of proteins by a peptide linker. Linker sequencewith regard to length, flexibility and hydrophilicity is important so asnot to disturb the functions of the domains. The linker regions of loop1 fusion constructs were initially designed to be of sufficient lengthto allow the correct folding of both the exonuclease and α-hemolysindomains of the fusion protein. However, of importance to the release ofmonophosphate nucleosides in a proximity to the pore lumen is the lengthand conformation of the linker regions. At some point, however, thelinkers will become too short to connect the subunits in their nativeconformation without strain, which may be particularly detrimental toexonuclease activity and probably oligomerisation. The length of thelinkers was therefore reduced to (SG)₄, (SG)₂ and (SG)₁ to determine theeffect on oligomerisation efficiency. For oligomerisation the shortened(SG)₄ and (SG)₂ linkers had no adverse effect on the efficiency ofheteroheptamer formation. The effect of these shortened linkers on theenzyme activity was not determined but the (SG)₄ fusion protein showedincreased expression of soluble protein, which is an indicator ofcorrectly folded proteins.

The conformational flexibility of these linkers will also have an effecton the exonuclease position in relation to the pore lumen at any giventime. While conformational flexibility may be required at the N andC-terminus linker juncture too much flexibility in the rest of thelinker may be detrimental to the co-localisation of the exonucleaseactive site to the pore lumen. The absence of a β-carbon in glycinepermits the polypeptide backbone to access dihedral angles that otheramino acids cannot. Proline, as a cyclic imino acid, has no amidehydrogen to donate in hydrogen bonding so cannot fit into either α-helixor β-strand secondary structure. Poly-proline regions are thereforestiff with the absence of secondary structure. By in vivo homologousrecombination of PCR products the 10 serine-glycine linker was replacedwith 5 proline residues. The use of a rigid polyproline “molecularrulers” was the determined for loop 1 EcoExoIII constructs as the linkerbetween the α-terminus of the exonuclease and the N-terminus ofα-hemolysin (FIG. 7).

Heteroheptamer formation was not abolished demonstrating the potentialuse of polyproline as a linker between the C-terminus of EcoExoIII andα-hemolysin T19 for the fusion protein. Although both fusion proteinsshowed a lower yield of heteroheptamers where the fusion protein ispredominant the formation in particular ofHL-(RQ)₆(RQC-EcoExoIII-L1-H6)₁ was unaffected.

The use of different length flexible linkers and alternative rigidlinkers for optimising the position and conformational freedom of theexonuclease in relation to the pore lumen, as well as a method foroptimising the formation of preferentially 6:1 heteroheptamers, has beendemonstrated.

2.2 Mutagenesis and Oligomerisation of Loop 2 Fusion Proteins

The high yield of heteroheptamers generated by IVTT proteins for theEcoExoIII in loop 1 gave confidence for insertion of EcoExoIII intoother loop regions, in particular both positions within loop 2 (FIG. 8).As this loop region connects two integral beta stands then it is likelythat any enzymes that do not have a co-localised N and C-terminus willbe too disruptive to the α-hemolysin domain, abolishing the ability ofthis protomer to oligomerise. Only very long linker regions may enablegenetic attachment of EcoExoI or TthRecJ at these positions, due totheir N and C-terminus localising to domains at distal ends of therespective enzymes.

The oligomerisation of the HL-RQC-EcoExoIII-L2a-H6 andHL-RQC-EcoExoIII-L2b-H6 fusion proteins was poor and only heptamers withan electrophoretic mobility similar to HL-(RQ)₇ andHL-(RQ)₆(RQC-EcoExoIII-L1-H6)₁ were observed. As oligomerisation ofHL-RQC-EcoExoIII-L2a-H6 was slightly improved over theHL-RQC-EcoExoIII-L2b-H6 fusion protein, modification was carried out toimprove the formation of heteroheptamer. Deletions of residues aroundthe insertion site were made in an attempt to accommodate the terminallinker residues. In addition certain residues in loop 2 may be importantfor heptamer self-assembly. Sequence alignment of the α-hemolysinmonomer with other β-pore forming toxin monomers, LukS and LukF,indicates loop 2 is a highly conserved region and in particular residueD45, which is the residue immediately after the exonuclease linkerjuncture. The crystal structure of the α-hemolysin heptamer alsoindicates that H48 is important to binding the amino latch of theadjoining subunit, at position T22 and D24 (Song, L., Hohaugh, M.,Shustak. C., Cheley, S., Bayley, H. and Gouaux, E. (1996). Structure ofStaphylococcal α-hemolysin, a heptameric transmembrane pore. Science.274, 1859-1865). Attempts to modify the insertion point to accommodateand characterise these potentially important interactions were thereforemade.

Around the loop 2a EcoExoIII insertion site (D44-D45) residues D45, K46and N47 were sequentially deleted by in vivo homologous recombination ofPCR products. To determine the importance of H48 the site of insertionwas also changed to lie between N47-N49, deleting H48 entirely. Aspreviously stated linker flexibility can have an important effect ofinteraction of domains within a fusion protein. Therefore the flexible10 serine glycine linkers were replaced with rigid 8 proline linkers inan attempt to confer greater domain separation. Each loop 2 fusionconstruct was expressed via IVTT and mixed in a 2.5:1 ratio withwild-type in the presence of purified rabbit red blood cell membranes.Any improvement in oligomerisation was determined by densitometry of theautoradiograph (FIG. 9).

Oligomerisation of the L2 fusion protein was abolished when theflexibility of the linker was changed to a more rigid polyprolinelinker. In addition deletion of H48 and positioning of the exonucleaseinsertion between N47 and N49 abolished heteroheptamer formation. Itappeared that only deletion of residues from around the D44-D45insertion site improved oligomerisation of the fusion protein. Todetermine if this could further be improved residue D45 was added backto the loop 2 deletion fusion proteins in a position adjacent to D44,before the EcoExoIII insertion site (FIG. 10).

Heteroheptamer formation was not affected by the position of residue D45and indeed adding back this residue to all fusion proteins wasdetrimental to oligomerisation, possibly as it reduced the number ofresidues deleted to accommodate the exonuclease by one as a consequence.Accommodating the exonuclease is therefore the key to improving theoligomerisation of the loop 2 fusion protein (as in SEQ ID NO: 26). Theinsertion site was varied further in an attempt to determine how closeto the β₂ strand the insertion site could be. The position within theloop region could be important for the relative positioning of theEcoExoIII active site in relation to the pore lumen and it is predictedthe closer to β₂ the better the presentation of cleaved monophosphatenucleosides. In each fusion construct the insertion site was not onlyvaried but the following three residues of α-hemolysin at the C-terminusof EcoExoIII were deleted in order to accommodate the exonuclease.Oligomerisation of the alternative loop 2 fusion proteinsHL-(RQ)₆(RQC-EcoExoIII-L2-D45-N47Δ-H6)₁.HL-(RQ)₆(RQC-EcoExoIII-L2-F42-D46Δ-H6)₁ andHL-(RQ)₆(RQC-EcoExoIII-L2-I43-D46Δ-H6)₁ determined that the insertionpoint can lie anywhere within the loop region but as soon as it breaks aregion of secondary structure all oligomerisation is abolished (FIG.10).

Whilst the linkers in the loop 2 fusion protein require some degree offlexibility, as determined by the fact that rigid polyproline linkerscould not substitute, the length can be reduced. The linker regions wereshortened as for the loop 1 EcoExoIII fusion protein to (SG)₄, (SG)₃,(SG)₂ and (SG)₁ to determine the effect on oligomerisation efficiency.For oligomerisation the shortened (SG)₄, (SG)₃ and (SG)₂ linkers had noadverse effect on the efficiency of heteroheptamer formation. The effectof these shortened linkers on the enzyme activity was not, however,determined.

2.3 Genetic Attachment at the N and C-Terminus of α-Hemolysin

Genetic attachment of two proteins, typically an enzyme to an antibody,has previously focused on the fusion of one protein's C-terminus toanother protein's N-terminus, mediated by a peptide linker. Aspreviously mentioned strategies for the attachment of a DNA handlingenzyme to the C or N-terminus of α-hemolysin was considered, inparticular the attachment of EcoExoI and the Klenow fragment. Attachmentof EcoExoI at the C-terminus was mediated by five different linkers inorder to determine the optimum fusion protein for oligomerisation. Asthe C-terminus is at the back of the α-hemolysin cap domain a turn ofapproximately 180° was desired. In order to initiate this turn either aGly-Asp or Trp-Pro-Val motif was added at the start of the linkerpeptide. Two linker peptides were also used, either a flexible 16serine-glycine or a 12 polyproline. As early results from the EcoExoIloop 1 fusion protein indicated that the 6:1 heteroheptamer had the sameelectrophoretic mobility as wild-type homoheptamer then a mixture ofradiolabelled and non-radio labelled IVTT monomers were used foroligomerisation. Monomers were mixed in a 1:1 ratio and oligomerised onpurified rabbit red blood cell membranes (FIG. 11).

Although the predominant fusion protein produced is the 6:1heteroheptamer this migrates to the same position as the HL-(RQ)₇homoheptamer. Therefore the proteins corresponding toHL-(RQ)₅(RQC-EcoExoI-Cter-{SG}8-H6)₂,HL-(RQ)₅(RQC-EcoExoI-Cter-DG{SG}8-H6)₂ as well as theHL-(RQ)₅(RQC-EcoExoI-L1-H6)₂ heteroheptamer from an earlier experimentwere purified from SDS and the ability to insert into planar lipidbilayers determined. All heteroheptamers were capable of inserting intothe lipid bilayer to give single channel recordings.

The success for fusion of the EcoExoI at the C-terminus of α-hemolysinmediated by an (SG)₈ and DG(SG)₈ peptide linker provides the method forthe later attachment of other DNA handling enzymes via genetic fusion,such as the Klenow fragment (SEQ ID NOs: 28 and 30). The advantages ofthe Klenow fragment are the fact it provides a molecular motor forstrand sequencing and also shows some resistance to SDS PAGE (Akeson,Personal Communication).

2.4 Non-SDS PAGE Purification of Heptamers

Sodium dodecyl sulphate (SDS) is an anionic surfactant that is highlydenaturing to proteins, due to its ability to disrupt non-covalent bondsand bind to the peptide chain. As existing heptamer purificationtechniques rely on the use of SDS PAGE then the effect of this detergenton EcoExoIII was determined by a fluorescence based activity assay (FIG.12, left panel).

Even a low concentration of SDS abolished EcoExoIII activity for thenative enzyme, making the classical SDS PAGE purification of heptamersdenaturing with regard to the exonuclease moiety of a fusion proteinheteroheptamer. An alternative purification method was developedtherefore using the alternative detergent, n-dodecyl-D-maltopyranoside(DDM). The effect of this surfactant on the EcoExoIII was determined andfound to be non-denaturing to the native enzyme (FIG. 12, right panel).Following oligomerisation on rabbit red blood cell membranes instead ofpurifying heptamers via SDS PAGE the lipid membranes were dissolved byaddition of 0.1% DDM for 15 minutes. Heteroheptamers were then purifiedaway from the wild-type homoheptamer by affinity purification to thehexa-His tag on the C-terminus of the fusion protein. A buffer exchangefurther removed any surfactant and heptamers were then used for singlechannel recordings. This method does not distinguish entirely betweenheteroheptamers so the formation of 5:2 was limited by optimising theratios of monomers mixed.

Purification via DDM extraction produced heptamers that showed anincreased number of blocking events and surfactant behaviour on thelipid bilayer in single channel recordings. Whilst the cause of thisinstability remains undetermined, it is likely to be a result of othermembrane proteins released from the rabbit red blood cell membranes,either affecting the lipid bilayer directly or else increasing theprotein associated surfactant carryover. Oligomerisation of α-hemolysinmonomers is classically facilitated either on purified rabbit red bloodcell membranes or deoxycholate micelles. The yield of heptamer fromdeoxycholate is too poor in this instance to be of use and as previouslymentioned the use of purified rabbit red blood cell membranes led tolipid bilayer instability. As an alternative, synthetic lipid vesicleswere developed based on the lipid composition of rabbit red blood cellmembranes, which lack other the membrane proteins of rabbit red bloodcell membranes. These are composed of 30% cholesterol, 30%phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), 10%sphingomyelin (SM) and 10% phosphatidylserine (PS). The synthetic lipidvesicles developed here give approximately the same efficiency ofheptamerisation as observed for rabbit red blood cell membranes.Heptamers purified from these synthetic lipid vesicles by DDM extractionalso showed a dramatic decrease in the occurrences of lipid bilayerinstability.

Oligomerisation and DDM purification of heptamers was also determinedfor E. coli expressed proteins. Expression of wild-type and fusionmonomers in E. coli gives a concentration sufficient for large scaleproduction of enzyme pores, typically 3 mg ml⁻¹ and 1 mg ml⁻¹respectively. Monomers were oligomerised on synthetic lipid vesicles ata ratio of 100:1 (wild-type:fusion) and purified as detailed previously(FIG. 13).

High level E. coli expression of monomers that can be oligomerised onsynthetic lipid vesicles was achieved. Purification of the 6:1heteroheptamer was also achieved in conditions that are non-denaturingto enzymes, ensuring activity of the pores exonuclease moiety.

2.5 Enzymatic Activity of Fusion Protein Heptamers

As the terminal ends of the enzyme are conformationally constrainedwithin loop regions of the α-hemolysin monomer then the dynamicmovements of the exonuclease domains necessary for activity could beimpacted. The native enzyme (Exonuclease III, NEB)) was able to cleavenucleotides from the dsDNA substrate to a point where the sense strandwas no longer of sufficient length to hybridise to the antisense strand(˜8 bp). On dissociation of the DNA strands the fluorophore, at the 5′end of the sense strand, was sufficiently spatially separated from itsquencher pair, at the 3′ end of the antisense strand, giving afluorescence increase relative to the enzyme activity. The activity ofthe native enzyme was also determined in a range of salt concentrations(0-1M KCl). Activity of the native enzyme was demonstrated inconcentrations ≤300 mM KCl, which is within the experimental conditionsrequired for single channel recordings and base discrimination. Todetermine if exonuclease activity of the EcoExoIII moiety on the fusionproteins was maintained after genetic attachment and oligomerisation,its activity was determined in this same fluorescence based DNAdegradation assay (FIG. 14).

The EcoExoIII fusion proteins demonstrated retained exonuclease activitybut as yet this is a qualitative rather than quantitative indication asamount of fusion protein was not determined. Therefore the effect ofgenetic fusion of the EcoExoIII to an α-hemolysin monomer on the rate ofexonuclease activity cannot be determined as yet.

The exonuclease activity of the fusion protein was checked at all stagesof purification and found to retain activity. Following oligomerisationand DDM purification the activity of fully formed pores was also checkedand found to show some exonuclease activity. This demonstrates theability to genetically couple an enzyme to a protein pore and stillretain activity of the enzyme after expression and oligomerisation to afully assembled pore.

2.6 Pore Forming Activity of Fusion Protein Heptamers.

As previously mentioned in the text the ability of a variety ofdifferent enzyme pore constructs to insert into lipid bilayers forsingle channel recordings has been shown. We have demonstrated thatchanges to the β-barrel of the α-hemolysin protein can enable covalentlinkage and stabilisation of an adapter molecule for continuous basedetection. For this the pore preferentially requires 6 subunits withmutations M113R/N139Q and 1 subunit with mutations M113R/N139Q/L135C. Todetermine if the exonuclease domain of the fusion protein within loopregions affected the ability of the pore to discriminate bases theM113R/N39Q/L135C mutations were made in the fusion constructs. As basediscrimination preferentially requires a heteroheptamer with only onesubunit carrying the L135C mutation and the enzyme pore preferentiallyone subunit being a fusion protein, the L135C mutation was made in thefusion protein. The wild-type M113R and N139Q construct from previouswork was used for the other subunits. E. coli expressed HL-RQ andHL-RQC-EcoExoIII-L2-D46-N47Δ-H6 were oligomerised on synthetic lipidvesicles (at a ratio of 100:1) and purified by DDM extraction. Theexonuclease activity of the fully formed pore was determined andindicated correct folding of the exonuclease moiety. The protein wasalso used for electrophysiology to determine firstly pore functionalityand secondly if base discrimination was possible (FIG. 19).

The 6:1 heteroheptamer can be inserted into a lipid bilayer and a stabletransmembrane current established. This current can be modulated by theintroduction of β-cyclodextrin, and is further reduced by the additionof monophosphate nucleosides. The presence of the exonuclease domainappears to have no detrimental effect on current flow or the basediscrimination by the pore. Although the work shown is for aheteroheptamer incorporating a fusion protein with the insertion ofEcoExoIII at the loop 2 position, similar data was acquired for the loop1 heteroheptamers.

Sequence listing SEQ ID NO: 1 1ATGGCAGATT CTGATATCAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CAGTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA141AAATCACAAT AAAAAACTGC TAGTTATTAG AACAAAAGGT ACCATTGCTG GTCAATATAG AGTTTATAGC211GAAGAAGGTG CTAACAAAAG TGGTTTAGCC TGGCCTTCAG CCTTTAAGGT ACAGTTGCAA CTACCTGATA281ATGAAGTAGC TCAAATATCT GATTACTATC CAAGAAATTC GATTGATACA AAAGAGTATA TGAGTACTTT351AACTTATGGA TTCAAGGGTA ATGTTACTGG TGATGATACA GGAAAAATTG GCGGCCTTAT TCGTGCAAAT421CTTTCCATTC GTCATACACT GAAATATGTT CAACCTGATT TCAAAACAAT TTTACACACC CCAACTCATA491AAAAAGTAGG CTGGAAAGTG ATATTTAACA ATATGGTGAA TCAAAATTGG GGACCATACG ATCGAGATTC561TTGGAACCCG GTATATGGCA ATCAACTTTT CATGAAAACT AGAAATGGTT CTATGAAAGC ACCAGATAAC631TTCCTTGATC CTAACAAAGC AAGTTCTCTA TTATCTTCAG GGTTTTCACC AGACTTCGCT ACAGTTATTA701CTATGGATAG AAAAGCATCC AAACAACAAA CAAATATAGA TGTAATATAC GAACGAGTTC GTGATGATTA771CCAATTGCAT TGGACTTCAA CAAATTGGAA AGGTACCAAT ACTAAAGATA AATGGACAGA TCGTTCTTCA841 GAAAGATATA AAATCGATTG GGAAAAAGAA GAAATGACAA AT SEQ ID NO: 2 1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE71EGANKSGLAW PSAFKVQLQL PDNEVAQISD YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV141SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR NGSMKAADNF211LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE281 RYKIDWEKEE MTN SEQ ID NO: 3 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA141AAATCACAAT AAAAAACTGC TAGTTATTAG AACAAAAGGT ACCATTGCTG GTCAATATAG AGTTTATAGC211GAAGAAGGTG CTAACAAAAG TGGTTTAGCC TGGCCTTCAG CCTTTAAGGT ACAGTTGCAA CTACCTGATA281ATGAAGTAGC TCAAATATCT GATTACTATC CAAGAAATTC GATTGATACA AAAGAGTATA GGAGTACTTT351AACTTATGGA TTCAACGGTA ATGTTACTGG TGATGATACA GGAAAAATTG GCGGCCTTAT TGGTGCACAA431CTTTCCATTC CTCATACACT CAAATATCTT CAACCTCATT TCAAAACAAT TTTACACACC CCAACTCATA491AAAAAGTAGG CTGGAAAGTG ATATTTAACA ATATGGTGAA TCAAAATTGG GGACCATACG ATCGAGATTC561TTGGAACCCG GTATATGGCA ATCAACTTTT CATGAAAACT AGAAATGGTT CTATGAAAGC AGCAGATAAC631TTCCTTGATC CTAACAAAGC AAGTTCTCTA TTATCTTCAG GGTTTTCACC AGACTTCGCT ACAGTTATTA701CTATGGATAG AAAAGCATCC AAACAACAAA CAAATATAGA TGTAATATAC GAACGAGTTC GTGATGATTA771CCAATTGCAT TGGACTTCAA CAAATTGGAA AGCTACCAAT ACTAAAGATA AATGGACACA TCCTTCTTCA841 GAAAGATATA AAATCGATTG GGAAAAAGAA GAAATGACAA AT SEQ ID NO: 4 1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE71EGANKSGLAW PSAFKVQLQL PDNEWAQISD YYPRNSIDTK EYRSTLTYGF NGNVTGDDTG KIGGLIGAQV141SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTE NGSMKAADNF211LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE281 RYKIDWEKEE MTN SEQ ID NO: 5 1TTGTTGAAGA CGAAAGGGCC TCGTGATACG CCTATTTTTA TAGGTTAATG TGATGATAAT AATGGTTTCT71TAGACGTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT141CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA GGAAGAGTAT211GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC281CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG351ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA421AGTTCTCCTA TGTGGCGCGG TATTATCCCG TGTTGACGCC GGCCAAGAGC AACTCGGTCG CCGCATACAC491TATTCTCAGA ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA561GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATCGG631AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA701CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGCAGCAATG GCAACAACGT771TGCGCAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC841GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA911GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG981TTATCTACAG GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC1051ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT1121TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT1191TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG1261CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA1331CCAAGTGTTT TTGCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC1401CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC1471AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG1541GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC1611TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC1681GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT1751AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC1821TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT1891CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA TACCGCTCGC1961CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA GCGCCTGATG CGGTATTTTC2031TCCTTACCCA TCTCTCCCCT ATTTCACACC CCATATATCC TCCACTCTCA CTACAATCTC CTCTCATCCC2101GCATAGTTAA GCCAGTATAC ACTCCGCTAT CGCTACGTGA CTGGGTCATG GCTGCGCCCC GACACCCGCC2171AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC2241TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGGCAGCGC TCTCCCTTAT2311GCGACTCCTG CATTAGGAAG CGACCCAGTA GTAGGTTGAG GCCGTTGAGC ACCGCCGCCG CAAGGAATGG2381TGCATGCAAG GAGATGGCGC CCAACAGTCC CCCGGCCACG GGGCCTGCCA CCATACCCAC GCCGAAACAA2451GCGCTGATGA GCCCGAAGTG GCGAGCCCGA TCTTCCCCAT CGGTGATGTC GGCGATATAG GCGCCAGCAA2521CCGCACCTGT GGCGCCGGTG ATGCCGGCCA CGATGCGTCC GGCGTAGAGG ATCGAGATCT AGCCCGCCTA3011TTACTATCCA AGAAATTCGA TTGATACAAA AGAGTATATG AGTACTTTAA CTTATGGATT CAACGGTAAT3081GTTACTGGTG ATGATACAGG AAAAATTGGC GGCCTTATTG GTGCAAATGT TTCGATTGGT CATACACTGA3151AATATGTTCA ACCTGATTTC AAAACAATTT TAGAGAGCCC AACTGATAAA AAAGTAGGCT GGAAAGTGAT3221ATTTAACAAT ATCGTGAATC AAAATTGGGG ACCATACGAT CCAGATTCTT CCAACCCCCT ATATCCCAAT3291CAACTTTTCA TGAAAACTAG AAATGGTTCT ATGAAAGCAG CAGATAACTT CCTTGATCCT AACAAAGCAA3361GTTCTCTATT ATCTTCAGGG TTTTCACCAG ACTTCGCTAC AGTTATTACT ATGGATAGAA AAGCATCCAA3431ACAACAAACA AATATAGATG TAATATACGA ACGAGTTCGT GATGATTACC AATTGCATTG GACTTCAACA3501AATTGGAAAG GTACCAATAC TAAAGATAAA TGGACAGATC GTTCTTCAGA AAGATATAAA ATCGATTGGG3571AAAAAGAAGA AATGACAAAT TAATGTAAAT TATTTGTACA TGTACAAATA AATATAATTT ATAACTTTAG3641CCGAAAGCTT GGATCCGGCT GCTAACAAAG CCCGAAAGGA AGCTGAGTTG GCTGCTGCCA CCGCTGAGCA3711ATAACTAGCA TAACCCCTTG GGGCCTCTAA ACGGGTCTTG AGGGGTTTTT TGCTGAAAGG AGGAACTATA3781TATAATTCGA GCTCGGTACC CACCCCGGTT GATAATCAGA AAAGCCCCAA AAACAGGAAG ATTGTATAAG3851CAAATATTTA AATTGTAAAC GTTAATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT3921GTTGTTCCAG TTTGGAACAA GAGTCCAGTA TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG3991GTTGTTCCAG TTTGGAACAA GAGTCCAGTA TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG4061TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCTA ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA4131AGCACTAAAT CGGAACCCTA AAGGGATGCC CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG4201AGAAAGCAAG GGAAGAAAGC CAAAGCAGCG GCCGCTAGCG CGCTCGCAAG TCTACCCCTC ACGCTGCGCG4271TAACCACCAC ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTGGGGA TCCTCTAGAG TCGACCTGCA4341GGCATGCAAG CTATCCCGCA AGAGGCCCGG CAGTACCGGC ATAACCAAGC CTATGCCTAC AGCATCCAGG4411GTGACGGTGC CGAGGATGAC GATGAGCGCA TTGTTAGATT TCATACACGG TGCCTGACTG CGTTAGCAAT4481TTAACTGTGA TAAACTACCG CATTAAAGCT AGCTTATCGA TGATAAGCTG TCAAACATGA GAASEQ ID NO: 6 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTTCC GGAACAGTAA71AAACAGGTGA TTTAGTCACT TATGATAAAG AAAATGGCAT GCACAAAAAA GTATTTTATA GTTTTATCGA141TGATAAAAAT CACAATAAAA AACTGCTAGT TATTAGAACA AAAGGTACCA TTGCTGGTCA ATATAGAGTT211TATAGCGAAG AAGGTGCTAA CAAAAGTGGT TTAGCCTGGC CTTCAGCCTT TAAGGTACAG TTGCAACTAC281CTGATAATGA AGTACCTCAA ATATCTGATT ACTATCCAAG AAATTCGATT CATACAAAAG AGTATATGAG351TACTTTAACT TATGGATTCA ACGGTAATGT TACTGGTGAT GATACAGGAA AAATTGGCGG CCTTATTGGT421GCAAATGTTT CGATTGGTCA TACACTGAAA TATGTTCAAC CTGATTTCAA AACAATTTTA GAGAGCCCAA491CTGATAAAAA AGTAGGCTGG AAAGTGATAT TTAACAATAT GGTGAATCAA AATTGGGGAC CATACGATCG561AGATTCTTGG AACCCGGTAT ATGGCAATCA ACTTTTCATG AAAACTAGAA ATGGTTCTAT GAAAGCAGCA631GATAACTTCC TTGATCCTAA CAAAGCAAGT TCTCTATTAT CTTCAGGGTT TTCACCAGAC TTCGCTACAG701TTATTACTAT GGATAGAAAA GCATCCAAAC AACAAACAAA TATAGATGTA ATATACGAAC GAGTTCGTGA771TGATTACCAA TTGCATTGGA CTTCAACAAA TTGGAAAGGT ACCAATACTA AAGATAAATG GACAGATCGT841 TCTTCAGAAA GATATAAAAT CGATTGGGAA AAAGAAGAAA TGACAAAT SEQ ID NO: 7 1ATGGCAGATT CTCATATTAA TATTAAAACC GGTACTACAG ATATTCGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATTCCGG141AGATAAAAAT CACAATAAAA AACTGCTAGT TATTAGAACA AAAGGTACCA TTGCTGGTCA ATATAGAGTT211TATAGCGAAG AACCTGCTAA CAAAACTCGT TTACCCTCCC CTTCACCCTT TAAGGTACAC TTGCAACTAC281CTGATAATGA AGTAGCTCAA ATATCTGATT ACTATCCAAG AAATTCGATT GATACAAAAG AGTATATGAG351TACTTTAACT TATGGATTCA ACGGTAATGT TACTGGTGAT GATACAGGAA AAATTGGCGG CCTTATTGGT421GCAAATGTTT CGATTGGTCA TACACTGAAA TATGTTCAAC CTGATTTCAA AACAATTTTA GAGAGCCCAA491CTGATAAAAA AGTAGGCTGG AAAGTGATAT TTAACAATAT GGTGAATCAA AATTGGGGAC CATACGATCG561AGATTCTTGG AACCCGGTAT ATGGCAATCA ACTTTTCATG AAAACTAGAA ATGGTTCTAT GAAAGCAGCA631CATAACTTCC TTCATCCTAA CAAAGCAAGT TCTCTATTAT CTTCAGGGTT TTCACCAGAC TTCGCTACAG701TTATTAGTAT GGATAGAAAA GCATCCAAAC AACAAACAAA TATAGATGTA ATATACGAAC GAGTTCGTGA771TGATTACCAA TTGCATTGGA CTTCAACAAA TTGGAAAGGT ACCAATACTA AAGATAAATG GACAGATCGT841 TCTTCAGAAA GATATAAAAT CGATTGGGAA AAAGAAGAAA TGACAAAT SEQ ID NO: 8 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA141AAATCACAAT AAATCCGGAA AACTGCTAGT TATTAGAACA AAAGGTACCA TTGCTGGTCA ATATAGAGTT211TATAGCGAAG AAGGTGCTAA CAAAAGTGGT TTAGCCTGGC CTTCAGCCTT TAAGGTACAG TTGCAACTAC281CTGATAATGA AGTAGCTCAA ATATCTGATT ACTATCCAAG AAATTCGATT GATACAAAAG AGTATATGAG351TACTTTAACT TATCCATTCA ACGGTAATGT TACTGGTCAT CATACACGAA AAATTCGCCG CCTTATTGGT421GCAAATGTTT CGATTGGTCA TACACTGAAA TATGTTCAAC CTGATTTCAA AACAATTTTA GAGAGCCCAA491CTGATAAAAA AGTAGGCTGG AAAGTGATAT TTAACAATAT GGTGAATCAA AATTGGGGAC CATACGATCG561AGATTCTTGG AACCCGGTAT ATGGCAATCA ACTTTTCATG AAAACTAGAA ATGGTTCTAT GAAAGCAGCA631GATAACTTCC TTGATCCTAA CAAAGCAAGT TCTCTATTAT CTTCAGGGTT TTCACCAGAC TTCGCTACAG701TTATTACTAT GGATAGAAAA GCATCCAAAC AACAAACAAA TATAGATGTA ATATACGAAC GAGTTCGTGA771TGATTACCAA TTGCATTGGA CTTCAACAAA TTGGAAAGGT ACCAATACTA AAGATAAATG GACAGATCGT841 TCTTCAGAAA GATATAAAAT CGATTGGGAA AAAGAAGAAA TGACAAAT SEQ ID NO: 9 1ATGAAATTTG TCTCTTTTAA TATCAACGGC CTGCGCGCCA GACCTCACCA GCTTGAAGCC ATCGTCGAAA71AGCACCAACG GGATGTGATT GGCCTGCAGG AGACAAAAGT TCATGACGAT ATGTTTCCGC TCGAAGAGGT141GGCGAAGCTC GGCTACAACG TGTTTTATCA CGGGCAGAAA GGCCATTATG GCGTGGCGCT GCTGACCAAA211GAGACGCCGA TTGCCGTGCG TCGCGGCTTT CCCGGTGACG ACGAAGAGGC GCAGCGGCGG ATTATTATGG281CGGAAATCCC CTCACTGCTG GGTAATGTCA CCGTGATCAA CGGTTACTTC CCGCAGGGTG AAAGCCGCGA351CGATGCGATA AAATTCCCGG CAAAAGCGCA GTTTTATCAG AATCTGCAAA ACTACCTGGA AACCGAACTC421AAACGTGATA ATCCGGTACT GATTATGGGC GATATGAATA TCAGCCCTAC AGATCTGGAT ATCGGCATTG491GCGAAGAAAA CCGTAAGCGC TGGCTGCGTA CCGGTAAATG CTCTTTCCTG CCGGAAGAGC GCGAATGGAT561GGACAGGCTG ATGAGCTGCG GGTTGGTCGA TACCTTCCGC CATGCGAATC CGCAAACAGC AGATCGTTTC631TCATGGTTTG ATTACCGCTC AAAAGGTTTT GACGATAACC GTGGTCTGCG CATCGACCTG CTGCTCGCCA710GCCAACCGCT GGCAGAATGT TGCGTAGAAA CCGGCATCGA CTATGAAATC CGCAGCATGG AAAAACCGTC771 CGATCACGCC CCCGTCTGCG CGACCTTCCG CCGC SEQ ID NO: 10 1MKPVSFNING LRARPHQLEA IVEKHQPDVI GLQETKVHDD MFPLEEVAKL GYNVFYHGQK GHYGVALLTK71ETPIAVRRGF PGDDEEAQRR IIMAEIPSLL GNVTVINGYF PQGESRDHPI KFPAKAQFYQ NLQNYLETEL141KRDNPVLIMG DMNISPTDLD IGIGEENRKR WLRTGKCSFL PERREWMDRL MSWGLVDTFR HANPQTADRF211 SWFDYRSKGF DDNRGLRIDL LLASQPLAEC CVETGIDYEI RSMEKPSDHA PVWATFRRSEQ ID NO: 11 1ATGATGAATG ACGGTAAGCA ACAATCTACC TTTTTGTTTC ACGATTACGA AACCTTTGGC ACGCACCCCG71CGTTAGATCG CCCTGCACAG TTCGCAGCCA TTCGCACCGA TAGCGAATTC AATGTCATCG GCGAACCCGA141AGTCTTTTAC TGCAAGCCCG CTGATGACTA TTTACCCCAG CCAGGAGCCG TATTAATTAC CGGTATTACC211CCGCAGGAAG CACGGGCGAA AGGAGAAAAC GAAGCCGCGT TTGCCGCCCG TATTCACTCG CTTTTTACCG281TACCGAAGAC CTGTATTCTG GGCTACAACA ATGTGCGTTT CGACGACGAA GTCACACGCA ACATTTTTTA351TCGTAATTTC TACGATCCTT ACGCCTGGAG CTGGCAGCAT GATAACTCGC GCTGGGATTT ACTGGATGTT421ATGCGTGCCT GTTATGCCCT GCGCCCGGAA GGAATAAACT GGCCTGAAAA TGATGACGGT CTACCGAGCT491TTCGCCTTGA GCATTTAACC AAAGCGAATG GTATTGAACA TAGCAACGCC CACGATGCGA TGGCTGATGT561GTACGCCACT ATTGCGATGG CAAAGCTGGT AAAAACGCGT CAGCCACGCC TGTTTGATTA TCTCTTTACC631CATCGTAATA AACACAAACT GATGGCGTTG ATTGATGTTC CGCAGATGAA ACCCCTGGTG CACGTTTCCG701GAATGTTTGG AGCATGGCGC GGCAATACCA GCTGGGTGGC ACCGCTGGCG TGGCATCCTG AAAATCGCAA771TCCCGTAATT ATCGTGGATT TGGCACGAGA CATTTCGCCA TTACTGGAAC TGGATAGCGA CACATTGCGC841GAGCGTTTAT ATACCGCAAA AACCGATCTT GGCGATAACG CCGCCGTTCC GGTTAAGCTG GTGCATATCA911ATAAATGTCC GGTGCTGGCC CAGGCGAATA CGCTACGCCC GGAAGATGCC GACCGACTGG GAATTAATCG981TCAGCATTGC CTCGATAACC TGAAAATTCT GCGTGAAAAT CCGCAAGTGC GCGAAAAAGT GGTGGCGATA1051TTCGCGGAAG CCGAACCGTT TACGCCTTCA GATAACGTGG ATGCACAGCT TTATAACGGC TTTTTCAGTG1121ACGCAGATCG TGCAGCAATG AAAATTGTGC TGGAAACCGA GCCGCGTAAT TTACCGGCAC TGGATATCAC1191TTTTGTTGAT AAACGGATTG AAAAGCTGTT GTTCAATTAT CGGGCACGCA ACTTCCCGGG GACGCTGGAT1261TATGCCGAGC AGCAACGCTG GCTGGAGCAC CGTCGCCAGG TCTTCACGCC AGAGTTTTTG CAGGGTTATG1331CTGATGAATT GCAGATGCTG GTACAACAAT ATGCCGATGA CAAAGAGAAA GCGGCGCTGT TAAAAGCACT1401 TTGGCAGTAG GCGGAAGAGA TTGTC SEQ ID NO: 12 1MMNDGKQQST FLFHDYETFG THPALDRPAQ FAAIRTDSEP NVIGEPEVFY CKPADDYLPQ PGAVLITGIT71PQEAPAKGEN EAAFAARIHS LFTVPKTCIL GYNNVRFDDE VTRNIFYRNF YDPYAWSWQH DNSRWDLLDV141MRACYALRPE GINWPENDDG LPSFRLEHLT KANGIEHSNA HDAMADVYAI IAMAKLVKTR QPRLFDYLFT211HRNKHKLMAL IDVPQMKPLV HVSGMFGAWR GNTSWVAPLA WHPENRNAVI MVDLAGDISP LLELDSDTLR281ERLYTAKTDL GDNAAVPVKL VHINKCPVLA QANTLRPEDA DRLGINRQHC LDNLKILREN PQVREKVVAI351FAEAEPFTPS DNVDAQLYNG FFSDADRAAM KIVLETEPRN LPALDITFVD KRIEKLLFNY RARNFDGTLD421 YAEQQRWLEH RRQVFTPEFL QGYADELQML VQQYADDKEK VALLKALWQY AEEIVSEQ ID NO: 13 1ATGTTTCGTG GTAAAGAAGA TCTGGATCCG CCGCTGGCAC TGCTGCCGCT GAAAGGCCTG CGCGAAGCCG71CCGCACTCCT GGAAGAAGCC CTGCGTCAAG GTAAACGCAT TCGTGTTCAC GCCGACTATG ATGCCGATGG141CCTGACCGGC ACCGCGATCC TGGTTCGTGG TCTGGCCGCC CTGGGTGCGG ATGTTCATCC GTTTATCCCG211CACCGCCTGG AAGAAGGCTA TGGTGTCCTG ATGGAACGCG TCCCGGAACA TCTGGAAGCC TCGGACCTGT281TTCTGACCGT TGACTGCCGC ATTACCAACC ATGCGGAACT GCGCGAACTG CTGGAAAATG GCGTGGAAGT351CATTGTTACC GATCATCATA CGCCGGGCAA AACGCCGCCG CCGGGTCTGG TCGTGCATCC GGCGCTGACG421CCGGATCTGA AAGAAAAACC GACCGGCGCA GGCGTGGCGT TTCTGCTGCT GTGGGCACTG CATGAACGCC491TGGGCCTGCC GCCGCCGCTG GAATACGCGG ACCTGGCAGC CGTTGGCACC ATTGCCGACG TTGCCCCGCT561GTGGGGTTGG AATCGTGCAC TGGTGAAAGA AGGTCTGGCA CGCATCCCGG CTTCATCTTG GGTGGGCCTG631CGTGTGCTGG CTGAAGCCGT GGGCTATACC GGCAAAGCGG TCGAAGTCGC TTTCCGCATC GCGCCGCGCA701TCAATGCCGC TTCCCGCCTG GGCGAAGCGG AAAAAGCCCT GCGCCTGCTG CTGACGGATG ATGCGGCAGA771AGCTCAGGCG CTGGTCGGCG AACTGCACCG TCTGAACGCC CGTCGTCAGA CCCTGGAAGA AGCGATGCTG841CGCAAACTGC TGCCGCAGGC CGACCCGGAA GCGAAAGCCA TCGTTCTGCT GGACCCGGAA GGCCATCCGG911GTGTTATGGG TATTGTGGCC TCTCGCATCC TGGAAGCGAC CCTGCGCCCG GTCTTTCTGG TGGCCCAGGG981CAAAGGCACC GTGCGTTCGC TGGCTCCGAT TTCCGCCGTC GAAGCACTGC GCAGCGCGGA AGATCTGCTG1051CTGCGTTATG GTGGTCATAA AGAAGCGGCG GGTTTCGCAA TGGATGAAGC GCTGTTTCCG GCGTTCAAAG1121CACGCGTTGA AGCGTATGCC GCACGTTTCC CGGATCCGGT TCGTGAAGTG GCACTGCTGG ATCTGCTGCC1191GGAACCGGGC CTGCTGCCGC AGGTGTTCCG TGAACTGGCA CTGCTGGAAC CGTATGGTGA AGGTAACCCG1261 GAACCGCTGT TCCTG SEQ ID NO: 14 1MFRRKEDLDP PLALLPLKCL REAAALLEEA LRQCKRIRVH CDYDADCLTC TAILVRCLAA LCADVHPFIP71HRLEEGYGVL MERVPEHLEA SDLFLTVDCG ITNHAELREL LENGVEVIVT DHHTPGKTPP PGLVVHPALT141PDLKERPTGA GVAFLLLWAL HERLGLPPPL EYADLAAVGT IADVAPLWGW NRALVKEGLA RIPASSWVGL211RLLAEAVGYT GKAVEVAFRI APRINAASRL GEAEKALRLL LTDDAAEAQA LVEGLHRLNA RRQTLEEAML281RKLLPQADPE AKAIVLLDPE GHPGVMGIVA SRILEATLRP VFLVAQGKGT VRSLAPISAV EALRSAEDLL351LRYGGHKEAA GFAMDEALFP ARKARVEAYA ARFPDPVREV ALLDLLPEPG LLPQVFRELA LLEPYGEGNP421 EPLFL SEQ ID NO: 15 1TCCGGAAGCG GCTCTGGTAG TGGTTCTGGC ATGACACCGG ACATTATCCT GCAGCGTACC GGGATCGATG71TGAGAGCTGT CGAACAGGGG GATGATGCGT GGCACAAATT ACGGCTCGGC GTCATCACCG CTTCAGAAGT141TCACAACGTG ATAGCAAAAC CCCGCTCCGG AAAGAAGTGG CCTGACATGA AAATGTCCTA CTTCCACACC211CTGCTTGCTG AGGTTTGCAC CGGTGTGGCT CCGGAAGTTA ACGCTAAAGC ACTGGCCTGG GGAAAACAGT281ACGAGAACGA CGCCAGAACC CTGTTTGAAT TCACTTCCGG CGTGAATGTT ACTGAATCCC CGATCATCTA351TGGCGACGAA AGTATGCGTA CCGCCTGCTC TCCCGATGGT TTATGCAGTG ACGGCAACGG CCTTGAACTG421AAATGCCCGT TTACCTCCCG GGATTTCATG AAGTTCCGGC TCGGTGGTTT CGAGGCCATA AAGTCAGCTT491ACATGGCCCA GGTGCAGTAC AGCATGTGGG TGACGCGAAA AAATGCCTGG TACTTTGCCA ACTATGACCC561GCGTATGAAG CGTGAAGGCC TGCATTATGT CGTGATTGAG CGGGATGAAA AGTACATGGC GAGTTTTGAC631GAGATCGTGC CGGAGTTCAT CGAAAAAATG GACGAGGCAC TGGCTGAAAT TGGTTTTGTA TTTGGGGAGC701 AATGGCGATC TGGCTCTGGT TCCGGCAGCG GTTCCGGA SEQ ID NO: 16 1MTPDIILQRT CIDVRAVEQG DDAWHKLRLG VITASEVHNV IAKPRSCKKW PDMKMSYFHT LLAEVCTCVA71PEVNAKALAW GKQYENDART LFEFTSGVNV TESPIIYRDE SMRTACSPDG LCSDGNGLEL KCPFTSRDFM141KFRLGGFEAT KSAYMAQVQY SMWVTRKNAW YFANYDPRMK REGLHYVVIE RDEKYMASFD EIVPEFIEKM211 DEALAEICFV FCEQWR  SEQ ID NO: 17 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTTCC GGAAGCGGCT71CTGGTAGTGG TTCTGGCATG AAATTTGTTA GCTTCAATAT CAACGGCCTG CGCGCGCGCC CGCATCAGCT141GGAAGCGATT GTGGAAAAAC ATCAGCCGGA TGTTATTGGT CTGCAGGAAA CCAAAGTTCA CGATGATATG211TTTCCGCTGG AAGAAGTGGC GAAACTGGGC TATAACGTCT TTTATCATGG CCAGAAAGGT CATTATGGCG281TGGCCCTGCT GACCAAAGAA ACCCCGATCG CGGTTCGTCG TGGTTTTCCG GGTGATGATG AAGAAGCGCA351GCGTCGTATT ATTATGGCGG AAATTCCGAG CCTGCTGGGC AATGTGACCG TTATTAACGG CTATTTTCCG421CAGGGCCAAA GCCGTGATCA TCCGATTAAA TTTCCGGCCA AAGCGCAGTT CTATCAGAAC CTGCAGAACT491ATCTGGAAAC CGAACTGAAA CGTCATAATC CGGTGCTGAT CATGGGCCAT ATGAACATTA GCCCGACCGA561TCTGGATATT GGCATTGGCG AAGAAAACCG TAAACGCTGG CTGCGTACCG GTAAATGCAG CTTTCTGCCG631GAAGAACGTG AATGGATGGA TCGCCTGATG AGCTGGGGCC TGGTGGATAC CTTTCGTCAT GCGAACCCGC701AGACCGCCGA TCGCTTTAGC TGGTTTGATT ATCGCAGCAA AGGTTTTGAT GATAACCGTG GCCTGCGCAT771TGATCTGCTG CTGGCGAGCC AGCCGCTGGC GGAATGCTGC GTTGAAACCG GTATTGATTA TGAAATTCGC841AGCATGCAAA AACCGAGCGA TCACGCCCCG GTGTGGGCGA CCTTTCGCCG CTCTGGCTCT GGTTCCGGCA911GGGGTTGCGG AACAGTAAAA ACAGGTGATT TAGTCACTTA TGATAAAGAA AATGGCATGC ACAAAAAAGT981ATTTTATAGT TTTATCGATG ATAAAAATCA CAATAAAAAA CTGCTAGTTA TTAGAACAAA AGGTACCATT1051GCTGGTCAAT ATAGAGTTTA TAGCGAAGAA GCTGCTAACA AAAGTGGTTT AGCCTGGCCT TCAGCCTTTA1121AGGTACAGTT GCAACTACCT GATAATGAAG TAGCTCAAAT ATCTGATTAC TATCCAAGAA ATTCGATTGA1191TACAAAAGAG TATATGAGTA CTTTAACTTA TGGATTCAAC GGTAATGTTA CTGGTGATGA TACAGGAAAA1261ATTGGCGGCC TTATTGGTGC AAATGTTTCG ATTGGTCATA CACTGAAATA TGTTCAACCT GATTTCAAAA1331CAATTTTAGA GAGCCCAACT GATAAAAAAG TAGGCTGGAA AGTGATATTT AACAATATGG TGAATCAAAA1401TTGGGGACCA TACGATCGAG ATTCTTGGAA CCCGGTATAT GGCAATCAAC TTTTCATGAA AACTAGAAAT1471CGTTCTATGA AACCAGCACA TAACTTCCTT CATCCTAACA AAGCAAGTTC TCTATTATCT TCACCCTTTT1541CACCAGACTT CGCTACAGTT ATTACTATGG ATAGAAAAGC ATCCAAACAA CAAACAAATA TAGATGTAAT1611ATACGAACGA GTTCGTGATG ATTACCAATT GCATTGGACT TCAACAAATT GGAAAGGTAC CAATACTAAA1681GATAAATGGA CAGATCGTTC TTCAGAAAGA TATAAAATCG ATTGGGAAAA AGAAGAAATG ACAAATGGTG1751 GTTCGGGCTC ATCTGGTGGC TCGAGTCACC ATCATCATCA CCAC SEQ ID NO: 18 1ADSDINIKTG TTDIGSNTSG SGSGSGSGMK FVSFNINGLR ARPHQLEAIV EKHQPDVIGL QETKVHDDMF71PLEEVAKLGY NVFYHGQKGH YGVALLTKET PIAVRRGPPG DDEEAQRRII MAEIPSLLGN VTVINGYFPQ141GESRDHPIKF PAKAQFYQNL QNYLETELKR DNPVLIMGDM NISFTDLDIG IGEENRKRWL RTGKCSFLPE211EREWMDRLMS WGLVDIFRHA NPQIADRFSW FDYRSKGFDD NRGLRIDLLL ASQPLAECCV EIGIDYEIRS281MEKPSDHAPV WATFRRSCSC SGSCSCTVKT GDLVTYDKEN GMHKKVFYSF IDDKNHNKKL LVIRTKGTIA351GQYRVYSEEG ANKSGLAWPS AFKVQLQLPD NEVAQISDYY PRNSIDTKEY MSTLTYGFNG NVTGDDTGKI421GGLIGANVSI GHTLKYVQPD FKTILESPTD KKVGWKVIFN NMVNQNWGPY DRDSWNPVYG NQLFMKTRNG491SMKAADNFLD PNKASSLLSS CFSPDFATVI TMDRKASKQQ TNIDVIYERV RDDYQLHWTS TNWKGTNTKD561 KWTDRSSERY KIDWEKEEMT NGGSGSSGGS SHHHHHH SEQ ID NO: 19 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTTCC GGAAGCGGCT71CTCCTAGTGG TTCTGGCATG AAATTTGTTA GCTTCAATAT CAACGCCCTG CGCGCGCGCC CGCATCAGCT141GGAAGCGATT GTGGAAAAAC ATCAGCCGGA TGTTATTGGT CTGCAGGAAA CCAAAGTTCA CGATGATATG211TTTCCGCTGG AAGAAGTGGC GAAACTGGGC TATAACGTGT TTTATCATGG CCAGAAAGGT CATTATGGCG281TGGCCCTGCT GACCAAAGAA ACCCCGATCG CGGTTCGTCG TGGTTTTCCG GGTGATGATG AAGAAGCGCA351GCGTCGTATT ATTATGGCGG AAATTCCGAG CCTGCTGGGC AATGTGACCG TTATTAACGG CTATTTTCCG421CAGGGCGAAA GCCGTGATCA TCCGATTAAA TTTCCGGCCA AAGCGCAGTT CTATCAGAAC CTGCAGAACT491ATCTGGAAAC CGAACTGAAA CGTGATAATC CGGTGCTGAT CATGGGCGAT ATGAACATTA GCCCGACCGA561TCTGGATATT GGCATTGGCG AAGAAAACCG TAAACGCTGG CTGCGTACCG GTAAATGCAG CTTTCTGCCG631GAAGAACGTG AATGGATGGA TCGCCTGATG AGCTGGGGCC TGGTGGATAC CTTTCGTCAT GCGAACCCGC701AGACCGCCGA TCGCTTTAGC TGGTTTGATT ATCGCAGCAA AGGTTTTGAT GATAACCGTG GCCTGCGCAT771TGATCTGCTG CTGGCGAGCC AGCCGCTGGC GGAATGCTGC GTTGAAACCG GTATTGATTA TGAAATTCGC841AGCATGGAAA AACCGAGCGA TCACGCCCCG GTGTGGGCGA CCTTTCGCCG CTCTGGCTCT GGTTCCGGCA911GCGGTTCCGG AACAGTAAAA ACAGGTGATT TAGTCACTTA TGATAAAGAA AATGGCATGC ACAAAAAAGT981ATTTTATAGT TTTATCGATG ATAAAAATGA CAATAAAAAA CTGCTAGTTA TTAGAACAAA AGGTACCATT1051GCTGGTCAAT ATAGAGTTTA TAGCGAAGAA GGTGCTAACA AAAGTGGTTT AGCCTGGCCT TCAGCCTTTA1121AGGTACAGTT GCAACTACCT GATAATGAAG TAGCTCAAAT ATCTGATTAC TATCCAAGAA ATTCGATTGA1191TACAAAAGAG TATAGGAGTA CTTTAACTTA TGGATTCAAC GGTAATGTTA CTGGTGATGA TACAGGAAAA1261ATTGGCGGCT GTATTGGTGC ACAAGTTTCG ATTGGTCATA CACTGAAATA TGTTCAACCT GATTTCAAAA1331CAATTTTACA CACCCCAACT CATAAAAAAC TACCCTCCAA ACTCATATTT AACAATATCC TCAATCAAAA1401TTGGGGACCA TACGATCGAG ATTCTTGGAA CCCGGTATAT GGCAATCAAC TTTTCATGAA AACTAGAAAT1471GGTTCTATGA AAGCAGCAGA TAACTTCCTT GATCCTAACA AAGCAAGTTC TCTATTATCT TCAGGGTTTT1541CACCAGACTT CGCTACAGTT ATTACTATGG ATAGAAAAGC ATCCAAACAA CAAACAAATA TAGATGTAAT1611ATACGAACGA GTTCGTGATG ATTACCAATT GCATTGGACT TCAACAAATT GGAAAGGTAC CAATACTAAA1681GATAAATGGA CAGATCGTTC TTCAGAAAGA TATAAAATCG ATTGGGAAAA AGAAGAAATG ACAAATGGTG1751 GTTCGGGCTC ATCTGGTGGC TCGAGTCACC ATCATCATCA CCAC SEQ ID NO: 20 1ADSDINIKTG TTDIGSNTSG SGSGSGSGMK FVSFNINGLR ARPHQLEAIV EKHQPDVIGL QETKVHDDMF71PLEEVAKLGY NVFYHGQKGH YGVALLTKET PTAVRRGFPG DDEEAQRRII MAEIPSLLGN VTVINGYFPQ141CESRDHPIKF PAKAQFYQNL QNYLETELKR DNPVLIMCDM NISPTDLDIC ICEENRKRWL RIGKCSFLPE211EREWMDRLMS WGLVDTFRHA NPQTADRFSW FDYRSKGFDD NRFLRIDLLL ASQPLAECCV ETGIDYEIRS281MEKPSDHAPV WATFRRSGSG SGSGSGTVKT GDLVTYDKEN GMHKKVPYSF IDDKNHNKKL LVIRTKGTIA351GQYRVYSEEG ANKSGLAWPS AFKVQLQLPD NEVAQISDYY PRNSIDTKEY RSTLTYGFNG NVTGDDTGKI421GGCIGAQVSI GHTLKYVQPD FKTILESPTD KKVGWKVIFN NMVNQNWGPY DRDSWNPVYG NQLFMKTRNG491SMKAADNFLD PNKASSLLSS GFSPDFAIVI TMDRKASKQQ TNIDVIYERV RDDYQLHWTS TNWKGTNTKD561 KWTDRSSERY KIDWEKEEMT NGGSGSSGGS SHHHHHH SEQ ID NO: 21 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTTCC GGAAGCGGCT71CTGGTAGTGG TTCTGGCATG ATGAACGATG GCAAACAGCA GAGCACCTTC CTGTTTCATG ATTATGAAAC141CTTCGGTACC CATCCGGCCC TGGATCCTCC GGCGCAGTTT GCGGCCATTC GCACCGATAG CGAATTCAAT211GTGATTGGCG AACCGGAAGT GTTTTATTGC AAACCGGCCG ATGATTATCT GCCGCAGCCG GGTGCGGTGC281TGATTACCGG TATTACCCCG CAGGAAGCGC GCGCGAAAGG TGAAAACGAA GCGGCGTTTG CCGCGCGCAT351TCATAGCCTG TTTACCGTGC CGAAAACCTG CATTCTGGGC TATAACAATG TGCGCTTCGA TGATGAAGTT 421ACCCGTAATA TCTTTTATCG TAACTTTTAT GATCCGTATG CGTGGAGCTG GCAGCATGAT AACAGCCGTT491GGGATCTGCT GGATGTGATG CGCGCGTGCT ATGCGCTGCG CCCGGAAGGC ATTAATTGGC CGGAAAACGA561TGATGGCCTG CCGAGCTTTC GTCTGGAACA TCTGACCAAA GCCAACGGCA TTGAACATAG CAATGCCCAT631GATGCGATGG CCGATGTTTA TGCGACCATT GCCATGGCGA AACTGGTTAA AACCCGTCAG CCGCGCCTGT701TTGATTATCT GTTTACCCAC CGTAACAAAC ACAAACTGAT GGCGCTGATT GATGTTCCGC AGATGAAACC771CCTGGTGCAT GTGAGCGGCA TGTTTGGCGC CTGGCGCGGC AACACCAGCT GGGTGGCCCC GCTGGCCTGG841CACCCGGAAA ATCGTAACGC CGTGATTATG GTTGATCTGG CCGGTGATAT TAGCCCGCTG CTGGAACTGG911ATAGCGATAC CCTGCGTGAA CGCCTGTATA CCGCCAAAAC CGATCTGGGC GATAATGCCG CCGTGCCGGT981GAAACTGGTT CACATTAACA AATGCCCGGT GCTGGCCCAG GCGAACACCC TGCGCCCGGA AGATGCGGAT1051CGTCTGGGTA TTAATCGCCA GCATTGTCTG GATAATCTGA AAATCCTGCG TGAAAACCCG CAGGTGCGTG1121AAAAAGTGGT GGCGATCTTC GCGGAAGCGG AACCGTTCAC CCCGAGCGAT AACGTGGATG CGCAGCTGTA1191TAACGGCTTC TTTAGCGATG CCGATCGCGC GGCGATGAAA ATCGTTCTGG AAACCGAACC GCGCAATCTG1261CCGGCGCTGG ATATTACCTT TGTTGATAAA CGTATTGAAA AACTGCTGTT TAATTATCGT GCGCGCAATT1331TTCCGGGTAC CCTGGATTAT GCCGAACAGC AGCGTTGGCT GGAACATCGT CGTCAGGTTT TCACCCCGGA1401ATTTCTGCAG GGTTATGCGG ATGAACTGCA GATGCTGGTT CAGCAGTATG CCGATGATAA AGAAAAAGTG1471GCGCTGCTGA AAGCGCTGTG GCAGTATGCG GAAGAAATCG TTTCTGGCTC TGGTTCCGGC AGCGGTTCCG1541GAACAGTAAA AACAGGTGAT TTAGTCACTT ATGATAAAGA AAATGGCATG CACAAAAAAG TATTTTATAG1611TTTTATCGAT GATAAAAATC ACAATAAAAA ACTGCTAGTT ATTAGAACAA AAGGTACCAT TGCTGGTCAA1681TATAGAGTTT ATAGCGAAGA AGGTGCTAAC AAAAGTGGTT TAGCCTGGCC TTCAGCCTTT AAGGTACAGT1751TGCAACTACC TGATAATGAA GTAGCTCAAA TATCTGATTA CTATCCAAGA AATTCGATTG ATACAAAAGA1821GTATAGGAGT ACTTTAACTT ATGGATTCAA CGGTAATGTT ACTGGTGATG ATACAGGAAA AATTGGCGGC1891TGTATTGGTG CACAAGTTTC GATTGGTCAT ACACTGAAAT ATGTTCAACC TGATTTCAAA ACAATTTTAG1961AGAGCCCAAC TGATAAAAAA GTAGGCTGGA AAGTGATATT TAACAATATG GTGAATCAAA ATTGGGGACC2031ATACGATCGA GATTCTTGGA ACCCGGTATA TGGCAATCAA CTTTTCATGA AAACTAGAAA TGGTTCTATG2101AAAGCAGCAG ATAACTTCCT TGATCCTAAC AAAGCAAGTT CTCTATTATC TTCAGGGTTT TCACCAGACT2171TCGCTACAGT TATTACTATG GATAGAAAAG CATCCAAACA ACAAACAAAT ATAGATGTAA TATACGAACG2241AGTTCGTGAT GATTACCAAT TGCATTGGAC TTCAACAAAT TGGAAAGGTA CCAATACTAA AGATAAATGG2311ACAGATCGTT CTTCAGAAAG ATATAAAATC GATTGGGAAA AAGAAGAAAT GACAAATGGT GGTTCGGGCT2381 CATCTGGTGG CTCGAGTCAC CATCATCATC ACCAC SEQ ID NO: 22 1ADSDINIKTG TTDIGSNTSG SGSGSGSGMM NDGKQQSTFL FHDYETFGTH PALDRPAQFA AIRTDSEFNV71IGEPEVFYCK PADDYLPQPG AVLITGITPQ EARAKGENEA AFAARIHSLF TVPKTCILGY NNVRFDDEVT141RNIFYRNFYD PYAWSWQHDN SRWDLLDVMR ACYALRPEGI NWPENDDGLP SFRLEHLTKA NGIEHSNAHD211AMADVYATIA MAKLVKTRQP RLFDYLFTHR NKHKLMALID VPQMKPLVHV SGMFGAWRGN TSWVAPLAWH281PENRNAVIMV DLAGDISPLL ELDSDTLRER LYTAKTDLGD NAAVPVKLVH INKCPVLAQA NTLRPEDADR351LGINRQHCLD NLKILRENPQ VREKVVAIFA EAEPFTPSDN VDAQLYNGFF SDADRAAMKI VLETEPRNLP421ALDITFVDKR IEKLLFNYRA RNFPGTLDYA EQQRWLEHRR QVFTPEPLQG YADELQMLVQ QYADDKEKVA491LLKALWQYAE EIVSGSGSGS GSGTVKTGDL VTYDKENGMH KKVFYSFIDD KNHNKKLLVI RTKGTIAGQY561RVYSEEGANK SGLAWPSAFK VQLQLPDNEV AQISDYYPRN SIDTKEYRST LTYGFNGNVT GDDTGKIGGC631IGAQVSIGHT LKYVQPDFKT ILESPTDKKV GWKVIFNNMV NQNWGPYDRD SWNPVYGNQL FMKTRNGSMK701AADNFLDPNK ASSLLSSGFS PDFATVITMD RKASKQQTNI DVIYERVRDD YQLHWTSTNW KGTNTKDKWT771 DRSSERYKID WEKEEMTNGG SGSSGGSSHH HHHH SEQ ID NO: 23 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTTCC GGAAGCGGCT71CTGGTAGTGG TTCTGGCATG TTTCGTCGTA AAGAAGATCT GGATCCGCCG CTGGCACTGC TGCCGCTGAA141AGGCCTGCGC GAAGCCGCCG CACTGCTGGA AGAAGCGCTG CGTCAAGGTA AACGCATTCG TGTTCACGGC211GACTATGATG CGGATGGCCT GACCGGCACC GCGATCCTGG TTCGTGGTCT GGCCGCCCTG GGTGCGGATG281TTCATCCGTT TATCCCGCAC CGCCTGGAAG AAGGCTATGG TGTCCTGATG GAACGCGTCC CGGAACATCT351CGAAGCCTCG CACCTGTTTC TGACCGTTGA CTGCGGCATT ACCAACCATG CGGAACTGCG CGAACTGCTG421GAAAATGGCG TGGAAGTCAT TGTTACCGAT CATCATACGC CGGGCAAAAC GCCGCCGCCG GGTCTGGTCG491TGCATCCGGC GCTGACGCCG GATCTGAAAG AAAAACCGAC CGGCGCAGGC GTGGCGTTTC TGCTGCTGTG561GGCACTGCAT GAACGCCTGG GCCTGCCGCC GCCGCTGGAA TACGCGGACC TGGCAGCCGT TGGCACCATT631GCCGACGTTG CCCCGCTGTG GGGTTGGAAT CGTGCACTGG TGAAAGAAGG TCTGGCACGC ATCCCGGCTT701CATCTTGGGT GGGCCTGCGT CTGCTGGCTG AAGCCGTGGG CTATACCGGC AAAGCGGTCG AAGTCGCTTT771CCGCATCGCG CCGCGCATCA ATGCGGCTTC CCGCCTGGGC GAAGCGGAAA AAGCCCTGCG CCTGCTGCTG841ACGGATGATG CGGCAGAAGC TCAGGCGCTG GTCGGCGAAC TGCACCGTCT GAACGCCCGT CGTCAGACCC911TGGAAGAAGC GATGCTGCGC AAACTGCTGC CGCAGGCCGA CCCGGAAGCG AAAGCCATCG TTCTGCTGGA981CCCGGAAGGC CATCCGGGTG TTATGGGTAT TGTGGCCTCT CGCATCCTGG AAGCGACCCT GCGCCCGGTC1051TTTCTGGTGG CCCAGGGCAA AGGCACCGTG CGTTCGCTGG CTCCGATTTC CGCCGTCGAA GCACTGCGCA1121GCGCGGAAGA TCTGCTGCTG CGTTATGGTG GTCATAAAGA AGCGGCGGGT TTCGCAATGG ATGAAGCGCT1191GTTTCCGGCG TTCAAAGCAC GCGTTGAAGC GTATGCCGCA CGTTTCCCGG ATCCGGTTCG TGAAGTGGCA1261CTGCTGGATC TGCTGCCGGA ACCGGGCCTG CTGCCGCAGG TGTTCCGTGA ACTGGCAGTG CTGGAACCGT1331ATGGTGAAGG TAACCCGGAA CCGCTCTTCC TCTCTGGCTC TGGTTCCGGC AGCGGTTCCG GAACACTAAA1401AACAGGTGAT TTAGTCACTT ATGATAAAGA AAATGGCATG CACAAAAAAG TATTTTATAG TTTTATCGAT1471GATAAAAATC ACAATAAAAA ACTGCTAGTT ATTAGAACAA AAGGTACCAT TGCTGGTCAA TATAGAGTTT1541ATAGCGAAGA AGGTGCTAAC AAAAGTGGTT TAGCCTGGCC TTCAGCCTTT AAGGTACAGT TGCAACTACC1611TGATAATGAA GTAGCTCAAA TATCTGATTA CTATCCAAGA AATTCGATTG ATACAAAAGA GTATAGGAGT1681ACTTTAACTT ATGGATTCAA CGGTAATGTT ACTGGTGATG ATACAGGAAA AATTGGCGGC TGTATTGGTG1751CACAAGTTTC GATTGGTCAT ACACTGAAAT ATGTTCAACC TGATTTCAAA ACAATTTTAG AGAGCCCAAC1821TGATAAAAAA GTAGGCTGGA AAGTGATATT TAACAATATG GTGAATCAAA ATTGGGGACC ATACGATCGA1891GATTCTTGGA ACCCGGTATA TGGCAATCAA CTTTTCATGA AAACTAGAAA TGGTTCTATG AAAGCAGCAG1961ATAACTTCCT TGATCCTAAC AAAGCAAGTT CTCTATTATC TTCAGGGTTT TCACCAGACT TCGCTACAGT2031TATTACTATG GATAGAAAAG CATCCAAACA ACAAACAAAT ATAGATGTAA TATACGAACG AGTTCGTGAT2101GATTACCAAT TGCATTGGAC TTCAACAAAT TGGAAAGGTA CCAATACTAA AGATAAATGG ACAGATCGTT2171CTTCAGAAAG ATATAAAATC GATTGGGAAA AAGAAGAAAT GACAAATGGT GGTTCGGGCT CATCTGGTGG2241 CTCGAGTCAG CATCATCATC ACCAG SEQ ID NO: 24 1ADSDINIKTG TTDIGSNTSG SGSGSGSGMF RRKEDLDPPL ALLPLKGLRE AAALLEEALR QGKRIRVHGD71YDADGLTGTA ILVRGLAALG ADVHPFIPHR LEFGYGVLME RVPEHLEASD LFLTVDCGIT NHAELRELLE141NGVEVIVTDH HTPGKTPPPG LVVHPALTPD LKEKPTGAGV AFLLLWALHE RLGLPPPLEY ADLAAVGTIA211DVAPLWGWNR ALVKEGLARI PASSWVGLRL LAEAVGYTGK AVEVAFRIAP RINAASRLGE AEKALRLLLT281DDAAEAQALV GELHRLNARR QTLEEAMLRK LLPQADPEAK AIVLLDPEGH PGVMGIVASR ILEATLRPVF351LVAQGKGTVR SLAPISAVEA LRSAEDLLLR YGGHKEAAGF AMDEALFPAF KARVEAYAAR FPDPVREVAL421LDLLPEPGLL PQVFRELALL EPYGEGNPEP LFLSGSGSGS GSGTVKTGDL VTYDKENGMH KKVFYSFIDD491KNHNKKLLVI RTKGTIAGQY RVYSEEGANK SGLAWPSAFK VQLQLPDNEV AQISDYYPRN SIDTKEYRST561LTYGFNGNVT GDDTGKIGGC IGAQVSIGHT LKYVQPDFKT ILESPTDKKV GWKVIFNNMV NQNWGPYDRD631SWNPVYGNQL FMKTRNCSMK AADNFLDPNK ASSLLSSCFS PDFATVITMD RKASKQQTNI DVIYERVRDD701 YQLHWTSTNW KGTNTKDKWT DRSSERYKID WEKEEMTNGG SGSSGGSSHH HHHHSEQ ID NO: 25 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATTCCGG141AAGCGGCTCT GGTAGTGGTT CTGGCATGAA ATTTGTTAGC TTCAATATCA ACGGCCTGCG CGCGCGCCCG211CATCAGCTGG AAGCGATTGT GGAAAAACAT CAGCCGGATG TTATTGGTCT GCAGGAAACC AAAGTTCACG281ATGATATGTT TCCGCTGGAA GAAGTGGCGA AACTGGGCTA TAACGTGTTT TATCATGGCC AGAAAGGTCA351TTATGGCGTG GCCCTGCTGA CCAAAGAAAC CCCGATCGCG GTTCGTCGTG GTTTTCCGGG TGATGATGAA421GAAGCGCAGC GTCGTATTAT TATGGCGGAA ATTCCGAGCC TGCTGGGCAA TGTGACCGTT ATTAACGGCT491ATTTTCCGCA GGGCGAAAGC CGTGATCATC CGATTAAATT TCCGGCCAAA GCGCAGTTCT ATCAGAACCT561GCAGAACTAT CTGGAAACCG AACTGAAACG TGATAATCCG GTGCTGATCA TGGGCGATAT GAACATTAGC631CCGACCGATC TGGATATTGG CATTGGCGAA GAAAACCGTA AACGCTGGCT GCGTACCGGT AAATGCAGCT701TTCTGCCGGA AGAACGTGAA TGGATGGATC GCCTGATGAG CTGGGGCCTG GTGGATACCT TTCGTCATCC771GAACCCGCAG ACCGCCGATC GCTTTAGCTG GTTTGATTAT CGCAGCAAAG GTTTTGATGA TAACCGTGGC841CTGCGCATTG ATCTGCTGCT GGCGAGCCAG CCGCTGGCGG AATGCTGCGT TGAAACCGGT ATTGATTATG911AAATTCCCAG CATGGAAAAA CCGAGCGATC ACGCCCCGGT GTGGGCGACC TTTCGCCGCT CTGGCTCTGG981TTCCGGCAGC GGTTCCGGAC ACAATAAAAA ACTGCTAGTT ATTAGAACAA AAGGTACCAT TGCTGGTCAA1051TATAGAGTTT ATAGCGAAGA AGGTGCTAAC AAAAGTGGTT TAGCCTGGCC TTCAGCCTTT AAGGTACAGT1121TGCAACTACC TGATAATGAA GTAGCTCAAA TATCTGATTA CTATCCAAGA AATTCGATTG ATACAAAAGA1191GTATAGGAGT ACTTTAACTT ATGGATTCAA CGGTAATGTT ACTGGTGATG ATACAGGAAA AATTGGCGGC1261TGTATTGGTG CACAAGTTTC GATTGGTCAT ACACTGAAAT ATGTTCAACC TGATTTCAAA ACAATTTTAG1331AGAGCCCAAC TGATAAAAAA GTAGGCTGGA AAGTGATATT TAACAATATG GTGAATCAAA ATTGGGGACC1401ATAGGATCGA GATTCTTGGA ACCCGGTATA TGGCAATCAA CTTTTCATGA AAACTAGAAA TGGTTCTATG1471AAAGCAGCAG ATAACTTCCT TGATCCTAAC AAAGCAAGTT CTCTATTATC TTCAGGGTTT TCACCAGACT1541TCGCTACAGT TATTACTATG GATAGAAAAG CATCCAAACA ACAAACAAAT ATAGATGTAA TATACGAACG1611AGTTCGTGAT GATTACCAAT TGCATTGGAC TTCAACAAAT TGGAAAGGTA CCAATACTAA AGATAAATGG1681ACAGATCGTT CTTCAGAAAG ATATAAAATC GATTGGGAAA AAGAAGAAAT GACAAATGGT GGTTCGGGCT1751 CATCTGGTGG CTCGAGTCAC CATCATCATC ACCAC SEQ ID NO: 26 1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDSGSGSG SGSGMKFVSP NINGLRARPH71QLEAIVEKHQ PDVIGLQETK VHDDMFPLEE VAKLGYNVFY HGQKGHYGVA LLTKETPIAV RRGFPGDDEE141AQRRIIMAEI PSLLGNVTVI NGYFPQGESR DHPIKFPAKA QFYQNLQNYL ETELKRDNPV LIMGDMNISP211TDLDIGIGEE NRKRWLRTGK CSFLPEEREW MDRLMSWGLV DTFRHANDQT ADRFSWFDYR SKGFDDNRGL281RIDLLLASQP LAECCVEIGI DYEIRSMEKP SDHAPVWATF RRSGSGSGSG SGHNKKLLVI RTKGTIAGQY351RVYSEECANK SCLAWPSAFK VQLQLPDNEV AQISDYYPRN SIDTKEYRST LTYCFNCNVT CDDTCKICCC421IGAQVSIGHT LKYVQPDFKT ILESPTDKKV GWKVIFNNMV NQNWGPYDRD SWNPVYGNQL FMKTRNGSMK491AADNFLDPNK ASSLLSSGFS PDFATVITMD RKASKQQTNI DVIYERVRDD YQLHWTSTNW KGTNTKDKWT561 DRSSERYKID WEKEEMTNGG SGSSGGSSHH HHHH SEQ ID NO: 27 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA141AAATCACAAT AAAAAACTGC TAGTTATTAG AACAAAAGGT ACCATTGCTG GTCAATATAG AGTTTATAGC211GAAGAAGGTG CTAACAAAAG TGGTTTAGCC TGGCCTTCAG CCTTTAAGGT ACAGTTGCAA CTACCTGATA281ATGAAGTAGC TCAAATATCT GATTACTATC CAAGAAATTC GATTGATACA AAAGAGTATA GGAGTACTTT351AACTTATGGA TTCAACGGTA ATGTTACTGG TGATGATACA GGAAAAATTG GCGGCTGTAT TGGTGCACAA421GTTTCGATTG GTCATACACT GAAATATGTT CAACCTGATT TCAAAACAAT TTTAGAGAGC CCAACTGATA491AAAAAGTAGG CTGGAAAGTG ATATTTAACA ATATGGTGAA TCAAAATTGG GGACCATACG ATCGAGATTC561TTGGAACCCG GTATATGGCA ATCAACTTTT CATGAAAACT AGAAATGGTT CTATGAAAGC AGCAGATAAC631TTCCTTGATC CTAACAAAGC AAGTTCTCTA TTATCTTCAG GGTTTTCACC AGACTTCGCT ACAGTTATTA701CTATGGATAG AAAAGCATCC AAACAACAAA CAAATATAGA TGTAATATAC GAACGAGTTC GTGATGATTA771CCAATTGCAT TGGACTTCAA CAAATTGGAA AGGTACCAAT ACTAAAGATA AATGGACAGA TCGTTCTTCA841GAAAGATATA AAATCGATTG GGAAAAAGAA GAAATGACAA ATTCCGGTAG CGGCTCTGGT TCTGGCTCTG911GTTCCGGCAG CGGTTCCGGA CAGAGCACCT TCCTGTTTCA TGATTATGAA ACCTTCGGTA CCCATCCGGC981CCTGGATCGT CCGGCGCAGT TTGCGGCCAT TCGCACCGAT AGCGAATTCA ATGTGATTGG CGAACCGGAA1051GTGTTTTATT GCAAACCGGC CGATGATTAT CTGCCGCAGC CGGGTGCGGT GCTGATTACC GGTATTACCC1121CGCAGGAAGC GCGCGCGAAA GGTGAAAACG AAGCGGCGTT TGCCGCGCGC ATTCATAGCC TGTTTACCGT1191GCCGAAAACC TGCATTCTGG GCTATAACAA TGTGCGCTTC GATGATGAAG TTACCCGTAA TATCTTTTAT1261CGTAACTTTT ATGATCCGTA TGCGTGGAGC TGGCAGCATG ATAACAGCCG TTGGGATCTG CTGGATGTGA1331TGCGCGCGTG CTATGCGCTG CGCCCGGAAG GCATTAATTG GCCGGAAAAC GATGATGGCC TGCCGAGCTT1401TCGTCTGGAA CATCTGACCA AAGCCAACGG CATTGAACAT AGCAATGCCC ATGATGCGAT GGCCGATGTT1471TATGCGACCA TTGCGATGGC GAAACTGGTT AAAACCCGTC AGCCGCGCCT GTTTGATTAT CTGTTTACCC1541ACCGTAACAA ACACAAACTC ATGGCCCTGA TTGATGTTCC GCACATCAAA CCGCTGCTGC ATGTGAGCGC1611CATGTTTGGC GCCTGGCGCG GCAACACCAG CTGGGTGGCC CCGCTGGCCT GGCACCCGGA AAATCGTAAC1681GCCGTGATTA TGGTTGATCT GGCCGGTGAT ATTAGCCCGC TGCTGGAACT GGATAGCGAT ACCCTGCGTG1751AACGCCTGTA TACCGCCAAA ACCGATCTGG GCGATAATGC CGCCGTGCCG GTGAAACTGG TTCACATTAA1821CAAATGCCCG GTGCTGGCCC AGGCGAACAC CCTGCGCCCG GAAGATGCGG ATCGTCTGGG TATTAATCGC1891CAGCATTGTC TGGATAATCT GAAAATCCTG CGTGAAAACC CGCAGGTGCG TGAAAAAGTG GTGGCGATCT1961TCGCGGAAGC GGAACCGTTC ACCCCGAGCG ATAACGTGGA TGCGCAGCTG TATAACGGCT TCTTTAGCGA2031TGCCGATCGC GCGGCGATGA AAATCGTTCT GGAAACCGAA CCGCGCAATC TGCCGGCGCT GGATATTACC2101TTTGTTGATA AAGGTATTGA AAAACTGCTG TTTAATTATC GTGCGCGCAA TTTTCCGGGT ACCCTGGATT2171ATGCCGAACA GCAGCGTTGG CTGGAACATC GTCGTCAGGT TTTCACCCCG GAATTTCTGC AGGGTTATGC2241GGATGAACTG CAGATGCTGG TTCAGCAGTA TGCCGATGAT AAAGAAAAAG TGGCGCTGCT GAAAGCGCTG2311 TGGCAGTATG CGGAAGAAAT CGTTTCTGGC TCTGGTCACC ATCATCATCA CCACSEQ ID NO: 28 1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE71EGANKSGLAW PSAFKVQLQL PDNEVAQISD YYPRNSIDTK EYRSTLTYGF NGNVTGDDTG KIGGCIGAQV141SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR NGSMKAADNF211LDDNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE281RYKIDWEKEE MTNSGSGSGS GSGSGSGSGQ STFLFHDYET FGTHPALDRP AQFAAIRTDS EFNVIGEPEV351FYCKPADDYL PQPGAVLITG ITPQEARAKG ENEAAFAARI HSLFTVPKTC ILGYNNVRFD DEVTRNIFYR421NFYDPYAWSW QHDNSRWDLL DVMRACYALR PEGINWPEND DGLPSFRLEH LTKANGIEHS NAHDAMADVY491ATIAMAKLVK TRQPRLFDYL FTHRNKHKLM ALIDVPQMKP LVHVSGMFGA WRGNTSWVAP LAWHPENRNA561VIMVDLAGDI SPLLELDSDT LRERLYTAKT DLGDNAAVPV KLVHINKCPV LAQANTLRPE DADRLGINRQ631HGLDNLKILR ENPQVREKVV AIFAEAEPFT PSDNVDAQLY NGFFSDADRA AMKIVLETEP RNLPALDITF701VDKRIEKLLF NYRARNFPGT LDYARQQRWL EHRRQVFTPR FLQGYADELQ MLVQQYADDK EKVALLKALW771 QYAEEIVSCS CHHHHHH SEQ ID NO: 29 1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA141AAATCACAAT AAAAAACTGC TAGTTATTAG AACAAAAGGT ACCATTGCTG GTCAATATAG AGTTTATAGC211GAAGAAGGTG GTAACAAAAG TGGTTTAGCC TGGCCTTCAG CCTTTAAGGT ACAGTTGCAA CTACCTGATA281ATGAAGTAGC TCAAATATCT GATTACTATC CAAGAAATTC GATTGATACA AAAGAGTATA GGAGTACTTT351AACTTATGGA TTCAACGGTA ATGTTACTGG TGATGATACA GGAAAAATTG GCGGCTGTAT TGGTGCACAA421GTTTCGATTG GTCATACACT GAAATATGTT CAACCTGATT TCAAAACAAT TTTAGAGAGC CCAACTGATA491AAAAAGTAGG CTGGAAAGTG ATATTTAACA ATATGGTGAA TCAAAATTGG GGACCATACG ATCGAGATTC561TTGGAACCCG GTATATGGCA ATCAACTTTT CATGAAAACT AGAAATGGTT CTATGAAAGC AGCAGATAAC631TTCCTTGATC CTAACAAAGC AAGTTCTCTA TTATCTTCAG GGTTTTCACC AGACTTCGCT ACAGTTATTA701CTATGGATAG AAAAGCATCC AAACAACAAA CAAATATAGA TGTAATATAC GAACGAGTTC GTGATGATTA771CCAATTGCAT TGGACTTCAA CAAATTGGAA AGGTACCAAT ACTAAAGATA AATGGACAGA TCGTTCTTCA841GAAAGATATA AAATCGATTG GGAAAAAGAA GAAATGACAA ATGATGGCTC CGGTAGCGGC TCTGGTTCTG911GCTCTGGTTC CGGCAGCGGT TCCGCACAGA GCACCTTCCT GTTTCATGAT TATGAAACCT TCGGTACCCA981TCCGGCCCTG GATCGTCCGG CGCAGTTTGC GGCCATTCGC ACCGATAGCG AATTCAATGT GATTGGCGAA1051CCGGAAGTGT TTTATTGCAA ACCGGCCGAT GATTATCTGC CGCAGCCGGG TGCGGTGCTG ATTACCGGTA1121TTACCCCGCA GGAAGCGCGC GCGAAAGGTG AAAACGAACC GGCGTTTGCC CCGCGCATTC ATACCCTGTT1191TACCGTGCCG AAAACCTGCA TTCTGGGCTA TAACAATGTG CGCTTCGATG ATGAAGTTAC CCGTAATATC1261TTTTATCGTA ACTTTTATGA TCCGTATGCG TGGAGCTGGC AGCATGATAA CAGCCGTTGG GATCTGCTGG1331ATGTGATGCG CGCGTGCTAT GCGCTGCGCC CGGAAGGCAT TAATTGGCCG GAAAACGATG ATGGCCTGCC1401GAGCTTTCGT CTGGAACATC TGACCAAAGC CAACGGCATT GAACATAGCA ATGCCCATGA TGCGATGGCC1471GATGTTTATG CGACCATTGC GATGGCGAAA CTGGTTAAAA CCCGTCAGCC GCGCCTGTTT GATTATCTGT1541TTACCCACCG TAACAAACAC AAACTGATGG CGCTGATTGA TGTTCCGCAG ATGAAACCGC TGGTGCATGT1611GAGCGGCATG TTTGGCGCCT GGCGCCGCAA CACCAGCTGG GTGGCCCCGC TGGCCTGGCA CCCGGAAAAT1681CGTAACGCCG TGATTATGGT TGATCTGGCC CGTGATATTA GCCCGCTGCT GGAACTGGAT AGCGATACCC1751TGCGTGAACG CCTGTATACC GCCAAAACCG ATCTGGGCGA TAATGCCGCC GTGCCGGTGA AACTGGTTCA1821CATTAACAAA TGCCCGGTGC TGGCCCAGGC GAACACCCTG CGCCCGGAAG ATGCGGATCG TCTGGGTATT1891AATCGCCAGC ATTGTCTGGA TAATCTGAAA ATCCTGCGTG AAAACCCGCA GGTGCGTGAA AAAGTGGTGG1961CGATCTTCGC GGAAGCGGAA CCGTTCACCC CGAGCGATAA CGTGGATGCG CAGCTGTATA ACGGCTTCTT2031TAGCGATGCC GATCGCGCGG CGATGAAAAT CGTTCTGGAA ACCGAACCGC GCAATCTGCC GGCGCTGGAT2101ATTACCTTTG TTGATAAACG TATTGAAAAA CTGCTGTTTA ATTATCGTGC CCGCAATTTT CCGGGTACCC2171TGGATTATGC CGAACAGCAG CGTTGGCTGG AACATCGTCG TCAGGTTTTC ACCCCGGAAT TTCTGCAGGG2241TTATGCGGAT GAACTGCAGA TGCTGGTTCA GCAGTATGCC GATGATAAAG AAAAAGTGGC GCTGCTGAAA2311 GCGCTGTGGC AGTATGCGGA AGAAATCGTT TCTGGCTCTG GTCACCATCA TCATCACCACSEQ ID NO: 30 1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE71EGANKSGLAW PSAFKVQLQL PDNEVAQISD YYPRNSIDTK EYRSTLTYGF NGNVTGDDTG KTGGCTGAQV141SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR NGSMKAADNF211LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE281RYKIDWEKEE MTNDGSGSGS GSGSGSGSGS GQSTFLFHDY ETFGTHPALD RPAQFAAIRT DSEFNVIGEP351EVFYCKPADD YLPQPGAVLI TGITPQEARA KGENEAAFAA RIHSLFTVPK TCILCYNNVR FDDEVTRNIF421YRNFYDPYAW SWQHDNSRWD LLDVMRACYA LRPEGINWPE NDDGLPSFRL EHLTKANGIE HSNAHDAMAD491VYATIAMAKL VKTRQPRLFD YLFTHRNKHK LMALIDVPQM KPLVHVSGMF GAWRGNTSWV APLAWHPENR561NAVIMVDLAC DISPLLELDS DTLRERLYTA KTDLCDNAAV PVKLVHINKC PVLAQANTLR PEDADRLCIN631RQHCLDNLKI LRENPQVREK VVAIFAEAEP FTPSDNVDAQ LYNGFFSDAD RAAMKIVLET EPRNLPALDI701TFVDKRIEKL LFNYRARNFP GTLDYAEQQR WLEHRRQVFT PEFLQGYADE LQMLVQQYAD DKEKVALLKA771 LWQYAEEIVS GSGHHHHHH

The invention claimed is:
 1. A method of attaching an enzyme to atransmembrane protein pore subunit comprising: (a) providing apreparation of a first fusion protein, wherein each of the first fusionproteins comprise (i) an enzyme and (ii) a first polypeptide linker,wherein one end of the first polypeptide linker is attached to thecarboxyl or amino terminus of the enzyme, and wherein the firstpolypeptide linker comprises an internal reactive lysine residue; (b)providing a preparation of a second fusion protein, wherein each of thesecond fusion proteins comprise (i) a transmembrane protein pore subunitand (ii) a second polypeptide linker, wherein one end of the secondpolypeptide linker is attached to the carboxyl or amino terminus of thetransmembrane protein pore subunit and wherein the second polypeptidelinker comprises an internal reactive amino acid group having a carboxylgroup that is specific for the reactive lysine in (a), and an affinitypurification tag; (c) performing a coupling reaction between the twofusion proteins of (a) and (b) above under suitable conditions such thatthe internal reactive lysine residue of the first linker covalentlyattaches to the internal reactive amino acid group of the second linker,thereby generating a population of enzyme-attached transmembrane proteinpore subunits; and (d) separating the preparation of enzyme-attachedtransmembrane protein pore subunits from any unreacted first fusionprotein and second fusion protein.
 2. A method according to claim 1wherein the first polypeptide linker is attached to the carboxylterminus of the enzyme.
 3. A method according to claim 1 wherein thesecond polypeptide linker is attached to the carboxyl terminus of thetransmembrane protein pore subunit.
 4. A method according to claim 1wherein the first polypeptide linker is attached to the carboxylterminus of the enzyme and the second polypeptide linker is attached tothe carboxyl terminus of the transmembrane protein pore subunit.
 5. Amethod according to claim 1 wherein the enzyme is a nucleic acidhandling enzyme.
 6. A method according to claim 1 wherein the enzyme isa polymerase, exonuclease, helicase or topoisomerase.
 7. A methodaccording to claim 1 wherein the enzyme is capable of catalyzing thesynthesis of a polymer.
 8. A method according to claim 1 wherein theenzyme is a DNA polymerase or is an RNA polymerase.
 9. A methodaccording to claim 1 wherein the transmembrane protein pore subunit isfrom a β-barrel pore or an α-helix pore.
 10. A method according to claim1 wherein the transmembrane protein pore subunit is from α-hemolysin.11. A method according to claim 5, wherein the enzyme retains theability to handle nucleic acids after being attached to thetransmembrane protein pore subunit.
 12. A method according to claim 7,wherein the enzyme retains the ability to catalyze synthesis of apolymer after being attached to the transmembrane protein pore subunit.13. A method according to claim 1, wherein the transmembrane proteinpore subunit retains its ability to form a pore after being attached tothe enzyme.
 14. The method of claim 1 further comprising, after step(d): (e) combining the preparation of enzyme-attached transmembraneprotein pore subunits with one or more wild-type transmembrane proteinpore subunits to produce heteroligomeric transmembrane protein poreshaving a desired stoichiometry of enzyme-attached transmembrane proteinpore subunits to wild-type subunits.
 15. The method according to claim14, wherein the heteroligomeric transmembrane protein pores comprise anα-hemolysin transmembrane protein pore subunit.
 16. The method accordingto claim 14, wherein the enzyme attached to the heteroligomerictransmembrane protein pores is an exonuclease or a polymerase enzyme.17. The method according to claim 14, wherein the one or more wild-typetransmembrane protein pore subunits are 5, 6, 7, or 8 wild-typetransmembrane protein pore subunits.
 18. The method of claim 14, whereinthe enzyme forms part of the cis side of the heteroligomerictransmembrane protein pores.
 19. A method of attaching an enzyme to atransmembrane protein pore subunit comprising: (a) providing apreparation of a first fusion protein, wherein each of the first fusionproteins comprise (i) transmembrane protein pore subunit and (ii) afirst polypeptide linker, wherein one end of the first polypeptidelinker is attached to the carboxyl or amino terminus of thetransmembrane protein pore subunit, and wherein the first polypeptidelinker comprises an internal reactive lysine residue; (b) providing apreparation of a second fusion protein, wherein each of the secondfusion proteins comprise (i) an enzyme and (ii) a second polypeptidelinker, wherein one end of the second polypeptide linker is attached tothe carboxyl or amino terminus of the enzyme and wherein the secondpolypeptide linker comprises an internal reactive amino acid grouphaving a carboxyl group that is specific for the internal reactivelysine in (a), and an affinity purification tag; (c) performing acoupling reaction between the two fusion proteins of (a) and (b) aboveunder suitable conditions such that the internal reactive lysine residueof the first linker covalently attaches to the internal reactive aminoacid group of the second linker, thereby generating a population ofenzyme-attached transmembrane protein pore subunits; and (d) separatingthe preparation of enzyme-attached transmembrane protein pore subunitsfrom any unreacted first fusion protein and second fusion protein. 20.The method of claim 19 further comprising, after step (d): (e) combiningthe preparation of enzyme-attached transmembrane protein pore subunitswith one or more wild-type transmembrane protein pore subunits toproduce heteroligomeric transmembrane protein pores having a desiredstoichiometry of enzyme-attached transmembrane protein pore subunits towild-type subunits.